Antibodies specific for native PrPsc

ABSTRACT

Antibodies are disclosed which specifically bind to native PrP Sc  in situ. Preferred antibodies bind only to the native PrP Sc  of a particular species e.g., human, cow, sheep, pig, etc. Particularly preferred antibodies bind specifically to a particular isoform of human PrP Sc . Preferred antibodies of the invention are (1) produced by phage display methodology, (2) bind specifically to native PrP Sc , (3) neutralizes the infectivity of prions, (4) bind to PrP Sc  in situ and (5) bind 50% or more of PrP Sc  in a liquid flowable sample. Antibodies of the invention can be bound to a substrate and used to assay a sample (which has any PrP c  de-natured via proteinase K) for the presence of PrP Sc  of a specific species which PrP Sc  is associated with disease. Antibodies which specifically bind to human PrP Sc  can be labeled and injected carrying out an in vivo diagnostic test to determine if the human is infected with prions associated with disease. The antibodies are preferably produced using phage display technology wherein the genetic material in the phage expressing the antibody is obtained from a mammal with an ablated endogenous PrP protein gene and an endogenous chimeric PrP gene which mammal had been inoculated with PrP Sc  to induce antibody production.

CROSS-REFERENCE

[0001] This application is a continuation-in-part of our earlier filedpending U.S. application Ser. No. 08/528,104, filed Sep. 14, 1995, whichapplication is incorporated herein by reference and to which applicationwe claim priority under 35 USC §120.

GOVERNMENT RIGHTS

[0002] The United States Government may have certain rights in thisapplication pursuant to Grant No. AGO 2132 awarded by the NationalInstitutes of Health.

FIELD OF THE INVENTION

[0003] This invention relates to methods for obtaining antibodies andassays for using such antibodies. More specifically, the inventionrelates to methods of obtaining antibodies which specifically bind tonaturally occurring forms of PrP^(Sc).

BACKGROUND OF THE INVENTION

[0004] Prions are infectious pathogens that cause central nervous systemspongiform encephalopathies in humans and animals. Prions are distinctfrom bacteria, viruses and viroids. The predominant hypothesis atpresent is that no nucleic acid component is necessary for infectivityof prion protein. Further, a prion which infects one species of animal(e.g., a human) will not infect another (e.g., a mouse).

[0005] A major step in the study of prions and the diseases that theycause was the discovery and purification of a protein designated prionprotein (“PrP”) [Bolton et al., Science 218:1309-11 (1982); Prusiner, etal., Biochemistry 21:6942-50 (1982); McKinley, et al., Cell 35:57-62(1983)]. Complete prion protein-encoding genes have since been cloned,sequenced and expressed in transgenic animals. PrP^(c) is encoded by asingle-copy host gene [Basler, et al., Cell 46:417-28 (1986)] and isnormally found at the outer surface of neurons. Prion diseases areaccompanied by the conversion of PrP^(c) into a modified form calledPrP^(Sc). However, the actual biological or physiological function ofPrP^(c) is not known.

[0006] The scrapie isoform of the prion protein (PrP^(Sc)) is necessaryfor both the transmission and pathogenesis of the transmissibleneurodegenerative diseases of animals and humans. See Prusiner, S. B.,“Molecular biology of prion disease,” Science 252:1515-1522 (1991). Themost common prion diseases of animals are scrapie of sheep and goats andbovine spongiform encephalopathy (BSE) of cattle [Wilesmith, J. andWells, Microbiol. Immunol. 172:21-38 (1991)]. Four prion diseases ofhumans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease(CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatalfamilial insomnia (FFI) [Gajdusek, D. C., Science 197:943-960 (1977);Medori et al., N. Engl. J. Med. 326:444-449 (1992)]. The presentation ofhuman prion diseases as sporadic, genetic and infectious illnessesinitially posed a conundrum which has been explained by the cellulargenetic origin of PrP.

[0007] Most CJD cases are sporadic, but about 10-15% are inherited asautosomal dominant disorders that are caused by mutations in the humanPrP gene [Hsiao et al., Neurology 40:1820-1827 (1990); Goldfarb et al.,Science 258:806-808 (1992); Kitamoto et al., Proc. R. Sac. Lond (Inpress) (1994)]. Iatrogenic CJD has been caused by human growth hormonederived from cadaveric pituitaries as well as dura mater grafts [Brownet al., Lancet 340:24-27 (1992)]. Despite numerous attempts to link CJDto an infectious source such as the consumption of scrapie infectedsheep meat, none has been identified to date [Harries-Jones et al., J.Neurol. Neurosurg. Psychiatry 51:1113-1119 (1988)] except in cases ofiatrogenically induced disease. On the other hand, kuru, which for manydecades devastated the Fore and neighboring tribes of the New Guineahighlands, is believed to have been spread by infection duringritualistic cannibalism [Alpers, M. P., Slow Transmissible Diseases ofthe Nervous System, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (NewYork: Academic Press), pp. 66-90 (1979)].

[0008] The initial transmission of CJD to experimental primates has arich history beginning with William Hadlow's recognition of thesimilarity between kuru and scrapie. In 1959, Hadlow suggested thatextracts prepared from patients dying of kuru be inoculated intonon-human primates and that the animals be observed for disease that waspredicted to occur after a prolonged incubation period [Hadlow, W. J.,Lancet 2:289-290 (1959)]. Seven years later, Gajdusek, Gibbs and Alpersdemonstrated the transmissibility of kuru to chimpanzees afterincubation periods ranging form 18 to 21 months [Gajdusek et al., Nature209:794-796 (1966)]. The similarity of the neuropathology of kuru withthat of CJD [Klatzo et al., Lab Invest. 8:799-847 (1959)] promptedsimilar experiments with chimpanzees and transmissions of disease werereported in 1968 [Gibbs, Jr. et al., Science 161:388-389 (1968)]. Overthe last 25 years, about 300 cases of CJD, kuru and GSS have beentransmitted to a variety of apes and monkeys.

[0009] The expense, scarcity and often perceived inhumanity of suchexperiments have restricted this work and thus limited the accumulationof knowledge. While the most reliable transmission data has been said toemanate from studies using non-human primates, some cases of human priondisease have been transmitted to rodents but apparently with lessregularity [Gibbs, Jr. et al., Slow Transmissible Diseases of theNervous System, Vol. 2, S. B. Prusiner and W. J. Hadlow, eds. (New York:Academic Press), pp. 87-110 (1979); Tateishi, et al., Prion Diseases ofHumans and Animals, Prusiner, et al., eds. (London: Ellis Horwood), pp.129-134 (1992)].

[0010] The infrequent transmission of human prion disease to rodents hasbeen cited as an example of the “species barrier” first described byPattison in his studies of passaging the scrapie agent between sheep androdents [Pattison, I. H., NINDB Monoqraph 2, D. C. Gajdusek, C. J. GibbsJr. and M. P. Alpers, eds. (Washington, D.C.: U.S. Government Printing),pp. 249-257 (1965)]. In those investigations, the initial passage ofprions from one species to another was associated with a prolongedincubation time with only a few animals developing illness. Subsequentpassage in the same species was characterized by all the animalsbecoming ill after greatly shortened incubation times.

[0011] The molecular basis for the species barrier between Syrianhamster (SHa) and mouse was shown to reside in the sequence of the PrPgene using transgenic (Tg) mice [Scott, et al., Cell 59:847-857 (1989)].SHaPrP differs from MoPrP at 16 positions out of 254 amino acid residues[Basler, et al., Cell 46:417-428 (1986); Locht, et al., Proc. Natl.Acad. Sci. USA 83:6372-6376 (1986)]. Tg(SHaPrP) mice expressing SHaPrPhad abbreviated incubation times when inoculated with SHa prions. Whensimilar studies were performed with mice expressing the human, or ovinePrP transgenes, the species barrier was not abrogated, i.e., thepercentage of animals which became infected were unacceptably low andthe incubation times were unacceptably long. Thus, it has not beenpossible, for example in the case of human prions, to use transgenicanimals (such as mice containing a PrP gene of another species) toreliably test a sample to determine if that sample is infected withprions. The seriousness of the health risk resulting from the lack ofsuch a test is exemplified below.

[0012] More than 45 young adults previously treated with HGH derivedfrom human pituitaries have developed CJD [Koch, et al., N. Engl. J.Med. 313:731-733 (1985); Brown, et al., Lancet 340:24-27 (1992);Fradkin, et al., JAMA 265:880-884 (1991); Buchanan, et al., Br. Med. J.302:824-828 (1991)]. Fortunately, recombinant HGH is now used, althoughthe seemingly remote possibility has been raised that increasedexpression of wtPrP^(c) stimulated by high HGH might induce priondisease [Lasmezas, et al., Biochem. Biophys. Res. Commun. 196:1163-1169(1993)]. That the HGH prepared from pituitaries was contaminated withprions is supported by the transmission of prion disease to a monkey 66months after inoculation with a suspect lot of HGH [Gibbs, Jr., et al.,N. Engl. J. Med. 328:358-359 (1993)]. The long incubation timesassociated with prion diseases will not reveal the full extent ofiatrogenic CJD for decades in thousands of people treated with HGHworldwide. Iatrogenic CJD also appears to have developed in fourinfertile women treated with contaminated human pituitary-derivedgonadotrophin hormone [Healy, et al., Br. J. Med. 307:517-518 (1993);Cochius, et al., Aust. N. Z. J. Med. 20:592-593 (1990); Cochius, et al.,J. Neurol. Neurosurg. Psychiatry 55:1094-1095 (1992)] as well as atleast it patients receiving dura mater grafts [Nisbet, et al., J. Am.Med. Assoc. 261:1118 (1989); Thadani, et al., J. Neurosurg. 69:766-769(1988); Willison, et al., J. Neurosurg. Psychiatric 54:940 (1991);Brown, et al., Lancet 340:24-27 (1992)]. These cases of iatrogenic CJDunderscore the need for screening pharmaceuticals that might possibly becontaminated with prions.

[0013] Recently, two doctors in France were charged with involuntarymanslaughter of a child who had been treated with growth hormonesextracted from corpses. The child developed Creutzfeldt-Jakob Disease.(See New Scientist, Jul. 31, 1993, page 4). According to the PasteurInstitute, since 1989 there have been 24 reported cases of CJD in youngpeople who were treated with human growth hormone between 1983 andmid-1985. Fifteen of these children have died. It now appears as thoughhundreds of children in France have been treated with growth hormoneextracted from dead bodies at the risk of developing CJD (see NewScientist, Nov. 20, 1993, page 10.) Prior attempts to create PrPmonoclonal antibodies have been unsuccessful (see Barry and Prusiner, J.of Infectious Diseases Vol. 154, No. 3, Pages 518-521 (1986). Thus thereis a need for an assay to detect compounds which result in disease.Specifically, there is a need for a convenient, cost-effective assay fortesting sample materials for the presence of prions which cause CJD. Thepresent invention offers such an assay.

SUMMARY OF THE INVENTION

[0014] Antibodies of the invention will specifically bind to a nativeprion protein (i.e., native PrP^(Sc)) in situ with a high degree ofbinding affinity. The antibodies can be placed on a substrate and usedfor assaying a sample to determine if the sample contains a pathogenicform of a prion protein. The antibodies are characterized by one or moreof the following features (1) an ability to neutralize infectiousprions, (2) will bind to prion proteins (PrP^(Sc)) in situ i.e., willbind to naturally occurring forms of a prion protein in a cell cultureor in vivo and without the need to treat (e.g., denature) the prionprotein, and (3) will bind to a high percentage of the PrP^(Sc) form(i.e. disease form) of prion protein in a composition e.g., will bind to50% or more of the PrP^(Sc) form of the prion proteins. Preferredantibodies are further characterized by an ability to (4) bind to aprion protein of only a specific species of mammals e.g., bind to humanprion protein and not prion protein of other mammals.

[0015] An important object is to provide antibodies which bind to nativeprion protein (PrP^(Sc)).

[0016] Another object is to provide antibodies which specifically bindto epitopes of prion proteins (PrP^(Sc)) of a specific species of animaland not to the prion protein (PrP^(Sc)) of other species of animals.

[0017] Another object is to provide monoclonal antibodies whichspecifically bind to prion proteins (PRPSc) associated with disease,(e.g., human PrP^(Sc)) which antibodies do not bind to denatured PrPproteins not associated with disease (e.g., human PrP^(c)).

[0018] Still another object is to provide specific methodology to allowothers to generate a wide range of specific antibodies characterized bytheir ability to bind one or more types of prion proteins from one ormore species of animals.

[0019] Another object of the invention is to provide an assay for thedetection of PrP^(Sc) forms of PrP proteins.

[0020] Another object of the invention is to provide an assay which canspecifically differentiate prion protein (PrP^(Sc)) associated withdisease from PrP^(Sc) not associated with disease.

[0021] Another object is to detect prions which specifically bind tonative PrP^(Sc) of a specific species such as a human, cow, sheep, pig,dog cat or chicken.

[0022] An advantage of the invention is that it provides a fast,efficient cost effective assay for detecting the presence of nativePrP^(Sc) in a sample.

[0023] A specific advantage is that the assay can be used as a screenfor the presence of prions (i.e., PrP^(Sc)) in products such aspharmaceuticals (derived from natural sources) food, cosmetics or anymaterial which might contain such prions and thereby provide furtherassurances as to the safety of such products.

[0024] Another advantage is that the antibodies which can be used with aprotease which denatures PrP^(c) thereby providing for a means ofdifferentiating between infectious (PrP^(Sc)) and non-infectious forms(PrP^(Sc)) of prions.

[0025] Yet another advantage of the invention is that antibodies of theinvention are characterized by their ability to neutralize theinfectivity of naturally occurring prions e.g., neutralize PrP^(Sc).

[0026] Another advantage is that antibodies of the invention will bindto (PrP^(Sc)) prion proteins in situ, i.e., will bind to naturallyoccurring (PrP^(Sc)) prions in their natural state in a cell culture orin vivo without requiring that the prion proteins be particularlytreated, isolated or denatured.

[0027] Another advantage is that the prion proteins of the inventionwill bind to a relatively high percentage of the infectious form of theprion protein (e.g., PrP^(Sc))—for example bind to 50% or more of thePrP^(Sc) form of prion proteins in a composition.

[0028] An important feature of the invention is that the methodologymakes it possible to create a wide variety of different prion proteinantibodies with the same or individually engineered features whichfeatures may make the antibody particularly suitable for uses such as(1) prion neutralization to purify a product, (2) the extraction ofprion proteins and (3) therapies.

[0029] A feature of the invention is that it uses phage displaylibraries in the creation of the antibodies.

[0030] Another feature of the invention is that the phage aregenetically engineered to express a specific binding protein of anantibody on their surface.

[0031] These and other objects, advantages, and features of theinvention will become apparent to those persons skilled in the art uponreading the details of the chimeric gene, assay method, and transgenicmouse as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 is a schematic view of a portion of PrP proteins showingthe differences between a normal, wild-type human PrP protein and anormal, wild-type mouse PrP protein;

[0033]FIG. 2 shows the amino acid sequence of mouse PrP along withspecific differences between mouse PrP and human PrP;

[0034]FIG. 3 shows the amino acid sequence of mouse PrP and specificallyshows differences between mouse PrP and bovine PrP;

[0035]FIG. 4 shows the amino acid sequence of mouse PrP and specificallyshows differences between mouse PrP and bovine PrP;

[0036]FIG. 5 is a bar graph of serum dilution vs optical density at 405nm for the mouse (D7282) for serum against denatured mouse PrP 27-30;

[0037]FIG. 6 shows the amino acid sequences of selected (A) heavy chainand (B) light chain variable regions generated by panning an IgG1library from mouse D7282 against denatured MoPrP 27-30 rods;

[0038]FIG. 7 shows the deduced amino acid sequences for some of thephage clones obtained in one panning against PrP;

[0039] FIGS. 8A-8H show photos of histoblots 8A, 8B, 8C, 8D, 8E, 8F, 8Gand 8H showing staining of SHaPrP 27-30 and denatured SHaPrP 27-30;

[0040]FIG. 9 is a graph showing the ELISA reactivity of purified Fabsagainst prion protein SHa 27-30;

[0041]FIG. 10 is a graph of the ELISA reactivity of purified Fabsagainst denatured prion protein SHa 27-30;

[0042]FIG. 11 is a photo showing amino precipitation of SHaPrP 27-30with recombinant Fabs of the invention; and

[0043]FIG. 12 is a photo showing amino precipitation of SHaPrP 27-30with purified Fabs of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0044] Before the present antibodies, assays and methods for producingan using such are disclosed and described, it is to be understood thatthis invention is not limited to particular antibodies, assays or methodas such may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

[0045] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

[0046] The terms “PrP protein”, “PrP” and the like are usedinterchangeably herein and shall mean both the infectious particle formPrP^(Sc) known to cause diseases (spongiform encephalopathies) in humansand animals and the non-infectious form PrP^(c) which, under appropriateconditions is converted to the infectious PrP^(Sc) form.

[0047] The terms “prion”, “prion protein” and “PrP^(Sc) protein” and thelike used interchangeably herein to refer to the infectious PrP^(Sc)form of a PrP protein and is a contraction of the words “protein” and“infection” and the particles are comprised largely if not exclusivelyof PrP^(Sc) molecules encoded by a PrP gene. Prions are distinct frombacteria, viruses and viroids. Known prions include those which infectanimals to cause scrapie, a transmissible, degenerative disease of thenervous system of sheep and goats as well as bovine spongiformencephalopathies (BSE) or mad cow disease and feline spongiformencephalopathies of cats. Four prion diseases known to affect humans are(1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familialinsomnia (FFI). As used herein prion includes all forms of prionscausing all or any of these diseases or others in any animals used—andin particular in humans and in domesticated farm animals.

[0048] The term “PrP gene” is used herein to describe genetic materialwhich expresses proteins as shown in FIGS. 2-4 and polymorphisms andmutations such as those listed herein under the subheading “PathogenicMutations and Polymorphisms.” The term “PrP gene” refers generally toany gene of any species which encodes any form of a prion protein. Somecommonly known PrP sequences are described in Gabriel et al., Proc.Natl. Acad. Sci. USA 89:9097-9101 (1992) which is incorporated herein byreference to disclose and de-scribe such sequences. The PrP gene can befrom any animal including the “host” and “test” animals described hereinand any and all polymorphisms and mutations thereof, it being recognizedthat the terms include other such PrP genes that are yet to bediscovered. The protein expressed by such a gene can assume either aPrP^(c) (non-disease) of PrP^(Sc) (disease) form.

[0049] The terms “standardized prion preparation”, “prion preparation”,“preparation” and the like are used interchangeably herein to describe acomposition containing prions (PrP^(Sc)) which composition is obtainedfrom brain tissue of mammals which contain substantially the samegenetic material as relates to prions, e.g., brain tissue from a set ofmammals which exhibit signs of prion disease which mammals (1) include atransgene as described herein; (2) have an ablated endogenous prionprotein gene; (3) have a high copy number of prion protein gene from agenetically diverse species; or (4) are hybrids with an ablatedendogenous prion protein gene and a prion protein gene from agenetically diverse species. The mammals from which standardized prionpreparations are obtained exhibit clinical signs of CNS dysfunction as aresult of inoculation with prions and/or due to developing the diseasedue to their genetically modified make up, e.g., high copy number ofprion protein genes.

[0050] The term “artificial PrP gene” is used herein to encompass theterm “chimeric PrP gene” as well as other recombinantly constructedgenes which when included in the genome of a host animal (e.g., a mouse)will render the mammal susceptible to infection from prions whichnaturally only infect a genetically diverse test mammal, e.g., human,bovine or ovine. In general, an artificial gene will include the codonsequence of the PrP gene of the mammal being genetically altered withone or more (but not all, and generally less than 40) codons of thenatural sequence being replaced with a different codon—preferably acorresponding codon of a genetically diverse mammal (such as a human).The genetically altered mammal being used to assay samples for prionswhich only infect the genetically diverse mammal. Examples of artificialgenes are mouse PrP genes encoding the sequence as shown in FIGS. 2, 3and 4 with one or more different replacement codons selected from thecodons shown in these Figures for humans, cows and sheep replacing mousecodons at the same relative position, with the proviso that not all themouse codons are replaced with differing human, cow or sheep codons.Artificial PrP genes can include not only codons of genetically diverseanimals but may include codons and codon sequences not associated withany native PrP gene but which, when inserted into an animal render theanimal susceptible to infection with prions which would normally onlyinfect a genetically diverse animal.

[0051] The terms “Ichimeric gene,” “chimeric PrP gene”, “chimeric prionproteins gene” and the like are used interchangeably herein to mean anartificially constructed gene containing the codons of a host animalsuch as a mouse with one or more of the codons being replaced withcorresponding codons from a genetically diverse test animal such as ahuman, cow or sheep. In one specific example the chimeric gene iscomprised of the starting and terminating sequence (i.e., N- andC-terminal codons) of a PrP gene of a mammal of a host species (e.g. amouse) and also containing a nucleotide sequence of a correspondingportion of a PrP gene of a test mammal of a second species (e.g. ahuman). A chimeric gene will, when inserted into the genome of a mammalof the host species, render the mammal susceptible to infection withprions which normally infect only mammals of the second species. Thepreferred chimeric gene disclosed herein is MHu2M which contains thestarting and terminating sequence of a mouse PrP gene and a non-terminalsequence region which is replaced with a corresponding human sequencewhich differs from a mouse PrP gene in a manner such that the proteinexpressed thereby differs at nine residues.

[0052] The term “genetic material related to prions” is intended tocover any genetic material which effects the ability of an animal tobecome infected with prions. Thus, the term encompasses any “PrP gene”,“artificial PrP gene”, “chimeric PrP gene” or “ablated PrP gene” whichterms are defined herein as well as modification of such which effectthe ability of an animal to become infected with prions. Standardizedprion preparations are produced using animals which all havesubstantially the same genetic material related to prions so that all ofthe animals will become infected with the same type of prions and willexhibit signs of infection at about the same time.

[0053] The terms “host animal” and “host mammal” are used to describeanimals which will have their genome genetically and artificiallymanipulated so as to include genetic material which is not naturallypresent within the animal. For example, host animals include mice,hamsters and rats which have their PrP gene ablated i.e., renderedinoperative. The host is inoculated with prion proteins to generateantibodies. The cells producing the antibodies are a source of geneticmaterial for making a phage library. Other host animals may have anatural (PrP) gene or one which is altered by the insertion of anartificial gene or by the insertion of a native PrP gene of agenetically diverse test animal.

[0054] The terms “test animal” and “test mammal” are used to describethe animal which is genetically diverse from the host animal in terms ofdifferences between the PrP gene of the host animal and the PrP gene ofthe test animal. The test animal may be any animal for which one wishesto run an assay test to determine whether a given sample contains prionswith which the test animal would generally be susceptible to infection.For example, the test animal may be a human, cow, sheep, pig, horse,cat, dog or chicken, and one may wish to determine whether a particularsample includes prions which would normally only infect the test animal.

[0055] The terms “genetically diverse animal” and “genetically diversemammal” are used to describe an animal which includes a native PrP codonsequence of the host animal which differs from the genetically diversetest animal by 17 or more codons, preferably 20 or more codons, and mostpreferably 28-40 codons. Thus, a mouse PrP gene is genetically diversewith respect to the PrP gene of a human, cow or sheep, but is notgenetically diverse with respect to the PrP gene of a hamster.

[0056] The terms “ablated PrP protein gene”, “disrupted PrP gene”, andthe like are used interchangeably herein to mean an endogenous PrP genewhich has been altered (e.g., add and/or remove nucleotides) in a mannerso as to render the gene inoperative. Examples of non-functional PrPgenes and methods of making such are disclosed in Bueler, H., et al“Normal development of mice lacking the neuronal cell-surface PrPprotein” Nature 356, 577-582 (1992) and Weisman (WO 93/10227). Themethodology for ablating a gene is taught in Capecchi, Cell 51:503-512(1987) all of which are incorporated herein by reference. Preferablyboth alleles of the genes are disrupted.

[0057] The terms “hybrid animal”, “transgenic hybrid animal” and thelike are used interchangeably herein to mean an animal obtained from thecross-breeding of a first animal having an ablated endogenous prionprotein gene with a second animal which includes either (1) a chimericgene or artificial PrP gene or (2) a PrP gene from a genetically diverseanimal. For example a hybrid mouse is obtained by cross-breeding a mousewith an ablated mouse gene with a mouse containing (1) human PrP genes(which may be present in high copy numbers) or (2) chimeric genes. Theterm hybrid includes any offspring of a hybrid including inbredoffspring of two hybrids provided the resulting offspring is susceptibleto infection with prions with normal infect only a genetically diversespecies. A hybrid animal can be inoculated with prions and serve as asource of cells for the creation of hybridomas to make monoclonalantibodies of the invention.

[0058] The terms “susceptible to infection” and “susceptible toinfection by prions” and the like are used interchangeably herein todescribe a transgenic or hybrid test animal which develops a disease ifinoculated with prions which would normally only infect a geneticallydiverse test animal. The terms are used to describe a transgenic orhybrid animal such as a transgenic mouse Tg(MHu2M) which, without thechimeric PrP gene, would not become infected with a human prion but withthe chimeric gene is susceptible to infection with human prions.

[0059] By “antibody” is meant an immunoglobulin protein which is capableof binding an antigen. Antibody as used herein is meant to include theentire antibody as well as any antibody fragments (e.g. F(ab′)², Fab′,Fab, Fv) capable of binding the epitope, antigen or antigenic fragmentof interest.

[0060] Antibodies of the invention are immunoreactive or immunospecificfor and therefore specifically and selectively bind to a PrP^(Sc)protein. Antibodies which are immunoreactive and immunospecific fornatural or native PrP^(Sc) are preferred. Antibodies for PrP^(Sc) arepreferably immunospecific—i.e., not substantially cross-reactive withrelated materials. Although the term “antibody” encompasses all types ofantibodies (e.g., monoclonal) the antibodies of the invention arepreferably produced using the phage display methodology describedherein.

[0061] By “purified antibody” is meant one which is sufficiently free ofother proteins, carbohydrates, and lipids with which it is naturallyassociated. Such an antibody “preferentially binds” to a native PrP^(Sc)protein (or an antigenic fragment thereof), i.e., does not substantiallyrecognize and bind to other antigenically-unrelated molecules. Apurified antibody of the invention is preferably immunoreactive with andimmunospecific for a PrP^(Sc) protein of specific species and morepreferably immunospecific for native human PrP^(Sc).

[0062] By “antigenic fragment” of a PrP protein is meant a portion ofsuch a protein which is capable of binding an antibody of the invention.

[0063] By “binds specifically” is meant high avidity and/or highaffinity binding of an antibody to a specific polypeptide i.e., epitopeof a PrP^(Sc) protein. Antibody binding to its epi-tope on this specificpolypeptide is preferably stronger than binding of the same antibody toany other epitope, particularly those which may be present in moleculesin association with, or in the same sample, as the specific polypeptideof interest e.g., binds more strongly to PrP^(Sc) than denaturedfragments of PrP^(c) so that by adjusting binding conditions theantibody binds almost exclusively to PrP^(Sc) and not denaturedfragments of PrP^(c). Antibodies which bind specifically to apolypeptide of interest may be capable of binding other polypeptides ata weak, yet detectable, level (e.g., 10% or less of the binding shown tothe polypeptide of interest). Such weak binding, or background binding,is readily discernible from the specific antibody binding to thecompound or polypeptide of interest, e.g. by use of appropriatecontrols. In general, antibodoies of the invention which bind to nativePrP^(Sc) in situ with a binding affinity of 10⁷ mole/l or more,preferably 10⁸ mole/liters or more are said to bind specifically toPrP^(Sc). In general, an antibody with a binding affinity of 10⁶mole/liters or less is not useful in that it will not bind an antigen ata detectable level using conventional methodology currently used.

[0064] By “detectably labeled antibody”, “detectably labeled anti-PrP”or “detectably labeled anti-PrP fragment” is meant an antibody (orantibody fragment which retains binding specificity), having an attacheddetectable label. The detectable label is normally attached by chemicalconjugation, but where the label is a polypeptide, it couldalternatively be attached by genetic engineering techniques. Methods forproduction of detectably labeled proteins are well known in the art.Detectable labels may be selected from a variety of such labels known inthe art, but normally are radioisotopes, fluorophores, paramagneticlabels, enzymes (e.g., horseradish peroxidase), or other moieties orcompounds which either emit a detectable signal (e.g., radioactivity,fluorescence, color) or emit a detectable signal after exposure of thelabel to its substrate. Various detectable label/substrate pairs (e.g.,horseradish peroxidase/diaminobenzidine, avidin/streptavidin,luciferase/luciferin)), methods for labelling antibodies, and methodsfor using labeled antibodies are well known in the art (see, forexample, Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)).

[0065] The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, particularly a human, andincludes:

[0066] (a) preventing the disease from occurring in a subject which maybe predisposed to the disease but has not yet been diagnosed as havingit;

[0067] (b) inhibiting the disease, i.e., arresting its development; or

[0068] (c) relieving the disease, i.e., causing regression of thedisease. The invention is directed toward treating patients withinfectious prions and is particularly directed toward treating humansinfected with PrP^(Sc), resulting in a disease of the central nervoussystem such as bovine spongiform encephalopathy; Creutzfeldt-JakobDisease; fatal familial insomnia or Gerstmann-Strassler-ScheinkerDisease.

[0069] Abbreviations used herein include:

[0070] CNS for central nervous system;

[0071] BSE for bovine spongiform encephalopathy;

[0072] CJD for Creutzfeldt-Jakob Disease;

[0073] FFI for fatal familial insomnia;

[0074] GSS for Gerstmann-Strassler-Scheinker Disease;

[0075] Hu for human;

[0076] HuPrP for a human prion protein;

[0077] Mo for mouse;

[0078] MoPrP for a mouse prion protein;

[0079] SHa for a Syrian hamster;

[0080] SHaPrP for a Syrian hamster prion protein;

[0081] Tg for transgenic;

[0082] Tg(SHaPrP) for a transgenic mouse containing the PrP gene of aSyrian hamster;

[0083] Tg(HuPrP) for transgenic mice containing the complete human PrPgene;

[0084] Tg(ShePrP) for transgenic mice containing the complete sheep PrPgene;

[0085] Tg(BovPrP) for transgenic mice containing the complete cow PrPgene;

[0086] PrP^(Sc) for the scrapie isoform of the prion protein;

[0087] PrP^(c) for the cellular contained comon, normal isoform of theprion protein;

[0088] MoPrP^(Sc) for the scrapie isoform of the mouse prion protein;

[0089] MHu2M for a chimeric mouse/human PrP gene wherein a region of themouse PrP gene is replaced by a corresponding human sequence whichdiffers from mouse PrP at 9 codons;

[0090] Tg(MHu2M) mice are transgenic mice of the invention which includethe chimeric MHu2M gene;

[0091] MHu2MPrP^(Sc) for the scrapie isoform of the chimeric human/mousePrP gene;

[0092] PrP^(CJD) for the CJD isoform of a PrP gene;

[0093] Prnp^(0/0) for ablation of both alleles of an endogenous prionprotein gene, e.g., the MoPrP gene;

[0094] Tg(SHaPrP^(+/0))81/Prnp^(0/0) for a particular line (81) oftransgenic mice expressing SHaPrP, +/0 indicates heterozygous;

[0095] Tg(HuPrP)/Prnp^(0/0) for a hybrid mouse obtained by crossing amouse with a human prion protein gene (HuPrP) with a mouse with bothalleles of the endogenous prion protein gene disrupted;

[0096] Tg(MHu2M)/Prnp^(0/0) for a hybrid mouse obtained by crossing amouse with a chimeric prion protein gene (MHu2M) with a mouse with bothalleles of the endogenous prion protein gene disrupted.

[0097] FVB for a standard inbred strain of mice often used in theproduction of transgenic mice since eggs of FVB mice are relativelylarge and tolerate microinjection of exogenous DNA relatively well.

General Aspect of the Invention

[0098] The core of the invention is an antibody which specifically bindsto a PrP^(Sc) protein and preferably binds to a native non-denaturedPrP^(Sc) protein in situ with an affinity of 10⁷ moles/liter or more,preferable 10⁸ moles/liter or more of a single species (e.g., human) andmore preferably binds only to human PrP^(Sc) and not denatured fragmentsof human PrP^(c)). The antibody may bind to all proteins coded by thedifferent mutations and/or polymorphisms of the PrP protein gene.Alternatively, a battery of antibodies (2 or more different antibodies)are provided wherein each antibody of the battery specifically binds toprotein coded by a different mutation or polymorphism of the PrP gene.The antibody can be bound to support surface and used to assay a samplein vitro for the presence of a particular type of human PrP^(Sc). Theantibody can also be bound to a detectable label and injected into ananimal to assay in vivo for the presence of a particular type of nativePrP^(Sc).

[0099] Although there are known procedures for producing antibodies fromany given antigen practice has shown that it is particularly difficultto produce antibodies which bind to certain proteins e.g., PrP^(Sc). Thedifficulty with obtaining antibodies to PrP^(Sc) relates, in part, toits special and unknown qualities. By following procedures describedherein antibodies which bind native PrP^(Sc) in situ have been obtainedand others may follow the procedures described here to obtain otherantibodies to PrP^(Sc) and to other proteins for which it is difficultto generate antibodies.

[0100] To produce antibodies of the invention it is preferable to beginwith inoculating a host mammal with prion proteins i.e., infectiousPrP^(Sc). The host mammal may be any mammal and is preferably a hostmammal of the type defined herein such as a mouse, rat, guinea pig orhamster and is most preferably a mouse. The host animal is inoculatedwith prion proteins which are endogenous to a different species which ispreferably a genetically diverse species. For example a mouse isinoculated with human prion proteins. Preferably, the host mammal isinoculated with infectious prion proteins of a genetically diversemammal. For example, a mouse is inoculated with human PrP^(Sc). Using anormal host mammal in this manner it is possible to elicit thegeneration of some antibodies. However, when a hosts animal includes aprion protein gene and is inoculated with prions from a geneticallydiverse species antibodies will, if at all, only be generated forepitopes which differ between epitopes of the prion protein of the hostanimal and epitopes of the genetically diverse species. Thissubstantially limits the amount of antibodies which might be generatedand decreases the ability to find an antibody which selectively binds toan infectious form of a prion protein and does not bind to denaturedfragments of a non-infectious form. Thus, unless one is attempting togenerate antibodies which differentiate between prion proteins ofdifferent species it is preferable to begin the antibody productionprocess using a mammal which has an ablated prion protein gene i.e., anull PrP gene abbreviated as Prnp^(0/0). Accordingly, the invention isgenerally described in connection with the use of such “null” mammalsand specifically described in connection with “null mice.”

[0101] Antibodies are produced by first producing a host animal (e.g., amouse) which has its endogenous PrP gene ablated, i.e., the PrP generendered inoperative. A mouse with an ablated PrP gene is referred to asa “null mouse”. A null mouse can be created by inserting a segment ofDNA into a normal mouse PrP gene and/or removing a portion of the geneto provide a disrupted PrP gene. The disrupted gene is injected into amouse embryo and via homologous recombination replaces the endogenousPrP gene.

[0102] The null mouse is injected with prions in order to stimulate theformation of antibodies. Further, injections of adjuvants and prions aregenerally used to maximize the generation of antibodies.

[0103] The mouse is then sacrificed and bone marrow and spleen cells areremoved. The cells are lysed, RNA is extracted and reversed transcribedto cDNA. Antibody heavy and light chains (or parts thereof) and thenamplified by PCR. The amplified cDNA library may be used as is or aftermanipulation to create a range of variants and thereby increase the sizeof the library.

[0104] An IgG phage display library is then constructed by inserting theamplified cDNA encoding IgG heavy chain and the amplified cDNA encodinga light chain into a phage display vector (e.g., a pComb3 vector) suchthat one vector contains a cDNA insert encoding a heavy chain fragmentin a first expression cassette of the vector, and a cDNA insert encodinga light chain fragment in a second expression cassette of the vector.

[0105] Ligated vectors are then packaged by filamentous phage M13 usingmethods well known in the art. The packaged library is then used toinfect a culture of E. coli, so as to amplify the number of phageparticles. After bacterial cell lysis, the phage particles are isolatedand used in a panning procedure.

[0106] The library created is panned against a composition containingprions. Antibody fragments which selectively bind to PrP^(Sc) e.g.,human PrP^(Sc) are then isolated.

Specifics of a PrP Protein

[0107] The major component of purified infectious prions, designated PrP27-30, is the proteinase K resistant core of a larger native proteinPrP^(Sc) which is the disease causing form of the ubiquitous cellularprotein PrP^(c). PrP^(Sc) is found only in scrapie infected cellswhereas PrP^(c) is present in both infected and uninfected cellsimplicating PrP^(Sc) as the major, if not the sole, component ofinfectious prion particles. Since both PrP^(c) and PrP^(Sc) are encodedby the same single copy gene, great effort has been directed towardunraveling the mechanism by which PrP^(Sc) is derived from PrP^(c).Central to this goal has been the characterization of physical andchemical differences between these two molecules. Propertiesdistinguishing PrP^(Sc) from PrP^(c) include low solubility (Meyer, etal 1986 PNAS), poor antigenicity (Kascack, J. Virol 1987; Serban D.1990) protease resistance (Oesch, et al 1985 Cell) and polymerization ofPrP 27-30 into rod-shaped aggregates which are very similar, on theultrastructural and histochemical levels, to the PrP amyloid plaquesseen in scrapie diseased brains (Prusiner, et al Cell 1983). By usingproteinase K it is possible to denature PrP^(c) but not PrP^(Sc). Todate, attempts to identify any post-transitional chemical modificationsin PrP^(c) that lead to its conversion to PrP^(Sc) have proven fruitless(Stahl, et al 1993 Biochemistry). Consequently, it has been proposedthat PrP^(c) and PrP^(Sc) are in fact conformational isomers of the samemolecule.

[0108] Conformational description of PrP using conventional techniqueshas been hindered by problems of solubility and the difficulty inproducing sufficient quantities of pure protein. However, PrP^(c) andPrP^(Sc) are conformationally distinct. Theoretical calculations basedupon the amino acid sequences of PrPs from several species havepredicted four putative helical motifs in the molecule. Experimentalspectroscopic data would indicate that in PrP^(c) these regions adoptα-helical arrangements, with virtually no β-sheet (Pan, et al PNAS1993). In dramatic contrast, in the same study it was found thatPrP^(Sc) and PrP 27-30 possess significant β-sheet content, which istypical of amyloid proteins. Moreover, studies with extended syntheticpeptides, corresponding to PrP amino acid residues 90-145, havedemonstrated that these truncated molecules may be converted to eitherα-helical or β-sheet structures by altering their solution conditions.The transition of PrP^(c) to PrP^(Sc) requires the adoption of β-sheetstructure by regions that were previously α-helical.

[0109] In general, scrapie infection fails to produce an immuneresponse, with host organisms being tolerant to PrP^(Sc) from the samespecies. Polyclonal anti-PrP antibodies have though been raised inrabbits following immunization with large amounts of SHaPrP 27-30(Bendheim, et al PNAS 1985, Bode, et al J. Gen. Virol. 1985). Similarly,a handful of anti-PrP monoclonal antibodies have been produced in mice(Kascack, et al, J. Virol. 1987, Barry, et al, J. Infect. Dis. 1986).These antibodies are able to recognize native PrP^(c) and denaturedPrP^(Sc) from both SHa and humans equally well, but do not bind toMoPrP. Unsurprisingly, the epitopes of these antibodies were mapped toregions of sequence containing amino acid differences between SHa- andMoPrP (Rogers, et al, J. Immunol. 1993).

[0110] It is not entirely clear as to why antibodies of the typedescribed in the above cited publications will bind to PrP^(c) but notto PrP^(Sc). Without being bound to any particular theory it issuggested that such may take place because epitopes which are exposedwhen the protein is in the PrP^(c) conformation are unexposed orpartially hidden in the PrP^(Sc) configuration—where the protein isrelatively insoluble and more compactly folded together. It is pointedout that stating that an antibody binds to PrP^(c) but not to PrP^(Sc)is not correct in absolute terms (but correct in commonly acceptedterms) because some minimal binding to PrP^(Sc) may occur. For purposesof the invention an indication that no binding occurs means that theequilibrium or affinity constant K_(a) is 10⁶ l/mole or less. Further,binding will be recognized as existing when the K_(a) is at 10⁷ l/moleor greater preferably 10⁸ l/mole or greater. The binding affinity of 10⁷l/mole or more may be due to (1) a single monoclonal antibody (i.e.,large numbers of one kind of antibodies) (2) a plurality of differentmonoclonal antibodies (e.g., large numbers of each of five differentmonoclonal antibodies) or (3) large numbers of polyclonal antibodies. Itis also possible to use combinations or (1)-(3).

[0111] Preferred antibodies will bind 50% or more of the PrP^(Sc) in asample. However, this may be accomplished by using several differentantibodies as per (1)-(3) above. It has been found that an increasednumber of different antibodies is more effective in binding a largerpercentage of the PrP^(Sc) in a sample as compared to the use of asingle antibody. For example, the use of six copies of a single antibody“Q” might bind 40% of the PrP^(Sc) in a sample. Similar results might beobtained with six copies of antibody “R” and “S”. However, by using twocopies each of “IQ”, “R” and “S” the six antibodies will bind over 50%of the PrP^(Sc) in a sample. Thus, a synergistic effect can be obtainedby combining combinations of two or more antibodies which bind PrP^(Sc)i.e., by combining two or more antibodies which have a binding affinityK_(a) for PrP^(Sc) of 10⁷ l/mole or more. Thus combination of D4, R2,6D2, D14, R1 and R10 and/or related antibodies can provide synergisticresults.

Antibody/Antigen Binding Forces

[0112] The forces which hold an antigen and antibody together are inessence no different from non-specific interactions which occur betweenany two unrelated proteins i.e., other macromolecules such as humanserum albumin and human transferrin. These intermolecular forces may beclassified into four general areas which are (1) electrostatic; (2)hydrogen bonding; (3) hydrophobic; and (4) Van der Waals. Electrostaticforces are due to the attraction between oppositely charged ionic groupson two protein side-chains. The force of attraction (F) is inverselyproportional to the square of the distance (d) between the charges.Hydrogen bonding forces are provided by the formation of reversiblehydrogen bridges between hydrophilic groups such as —OH, —NH₂ and —COOH.These forces are largely dependent upon close positioning of twomolecules carrying these groups. Hydrophobic forces operate in the sameway that oil droplets in water merge to form a single large drop.Accordingly, non-polar, hydrophobic groups such as the side-chains onvaline, leucine and phenylalanine tend to associate in an aqueousenvironment. Lastly, Van der Waals are forces created between moleculeswhich depend on interaction between the external electron clouds.

[0113] Further information regarding each of the different types offorces can be obtained from “Essential Immunology” edited by I. M.Roitti (6th Edition) Blackwell Scientific Publications, 1928. Withrespect to the present invention useful antibodies exhibit all of theseforces. It is by obtaining an accumulation of these forces in largeramounts that it is possible to obtain an antibody which has a highdegree of affinity or binding strength to the PrP protein and inparticular an antibody which has a high degree of binding strength toPrP^(Sc) in situ.

Measuring Antibody/Antigen Binding Strength

[0114] The binding affinity between an antibody and an antigen can bemeasured which measurement is an accumulation of a measurement of all ofthe forces described above. Standard procedures for carrying out suchmeasurements exist and can be directly applied to measure the affinityof antibodies of the invention for PrP proteins including nativePrP^(Sc) in situ.

[0115] One standard method for measuring antibody/antigen bindingaffinity is through the use of a dialysis sac which is a containercomprised of a material which is permeable to the antigen butimpermeable to the antibody. Antigens which are bound completely orpartially to antibodies are placed within the dialysis sac in a solventsuch as in water. The sac is then placed within a larger container whichdoes not contain antibodies or antigen but contains only the solvente.g., the water. Since only the antigen can diffuse through the dialysismembrane the concentration of the antigen within the dialysis sac andthe concentration of the antigen within the outer larger container willattempt to reach an equilibrium. After placing the dialysis sac into thelarger container and allowing for time to pass towards reaching anequilibrium it is possible to measure the concentration of the antigenwithin the dialysis sac and within the surrounding container and thendetermine the differences in concentration. This makes it possible tocalculate the amount of antigen which remains bound to antibody in thedialysis sac and the amount which disassociates from the antibody anddiffuses into the surrounding container. By constantly renewing thesolvent (e.g., the water) within the surrounding container so as toremove any antigen which is diffused thereinto it is possible to totallydisassociate the antibody from antigen within the dialysis sac. If thesurrounding solvent is not renewed the system will reach an equilibriumand it is possible to calculate the equilibrium constant (K) of thereaction i.e., the association and disassociation between the antibodyand antigen. The equilibrium constant (K) is calculated as an amountequal to the concentration of antibody bound to antigen within thedialysis sac divided by the concentration of free antibody combiningsites times the concentration of free antigen. The equilibrium constantor “K” value is generally measured in terms of liters per mole. The Kvalue is a measure of the difference in free energy (deta g) between theantigen and antibody in the free state as compared with the complexedform of the antigen and antibody. When using the phage displaymethodology described below the antibodies obtained have an affinity orK value of 10⁷ mole/liter or more.

Antibody Avidity

[0116] As indicated above the term “affinity” describes the binding ofan antibody to a single antigen determinate. However, in most practicalcircumstances one is concerned with the interaction of an antibody witha multivalent antigen. The term “avidity” is used to express thisbinding. Factors which contribute to avidity are complex and include theheterogeneity of the antibodies in a given serum which are directedagainst each determinate on the antigen and the heterogeneity of thedeterminants themselves. The multivalence of most antigens leads to aninteresting “bonus” effect in which the binding of two antigen moleculesby an antibody is always greater, usually many fold greater, than thearithmetic sum of the individual antibody links. Thus, it can beunderstood that the measured avidity between an antiserum and amultivalent antigen will be somewhat greater than the affinity betweenan antibody and a single antigen determinate.,

Null PrP Mice to make Antibodies

[0117] The present invention circumvents problems of tolerance and moreefficiently generates panels of monoclonal antibodies capable ofrecognizing diverse epitopes on Mo and other PrPs in part using micewith both alleles of the PrP gene, (Prnp) are ablated (Prnp^(0/0))(Bueler, et al, 1992). These PrP-deficient mice (or null mice), areindistinguishable from normal mice in their development and behavior.These null mice are resistant to scrapie following intracerebralinoculation of infectious MpPrP^(Sc) (Bueler, et al, 1993 Cell;Prusiner, et al, PNAS 1993). In addition Prnp^(0/0) mice will developIgG serum titers against Mo-, SHa and human PrP following immunizationwith relatively small quantities of purified SHaPrP 27-30 in adjuvant(Prusiner, et al, PNAS 1993). After allowing sufficient time to generateantibodies the immunized Prnp^(0/0) mice were sacrificed for hybridomaproduction in the conventional manner. Fusions derived from these micedid secrete PrP specific antibody. However, these hybridomas would notsecrete PrP specific antibodies for more than a few hours. In view ofthe somewhat limited success a different approach was taken.

Phage Display

[0118] Combinatorial antibody library technology, i.e., antigen basedselection from antibody libraries expressed on the surface of M13filamentous phage, offers a new approach to the generation of monoclonalantibodies and possesses a number of advantages relative to hybridomamethodologies which are particularly pertinent to the prion problem(Huse, et al, 1989; Barbas, et al, 1991: Clackson, et al. 1991; Burtonand Barbas, 1994). The present invention uses such technology to providePrP-specific monoclonal antibodies from phage antibody librariesprepared from MoPrP-immunized Prnp^(0/0) mice. The invention providesthe first monoclonal antibodies recognizing MoPrP in situ anddemonstrates the application of combinatorial libraries for cloningspecific antibodies from null mice. The general methodologies involvedin creating large combinatorial libraries using phage display technologyis described and disclosed in U.S. Pat. No. 5,223,409 issued Jun. 29,1993 which patent is incorporated herein by reference to disclose anddescribe phage display methodology.

Null Animals

[0119] The invention is largely described herein with respect to nullmice i.e., FVB mice with both alleles of the PrP gene ablated. However,other host animals can be used and preferred host animals are mice andhamsters, with mice being most preferred in that there existsconsiderable knowledge on the production of transgenic animals. Possiblehost animals include those belonging to a genus selected from Mus (e.g.mice), Rattus (e.g. rats), Oryctolagus (e.g. rabbits), and Mesocricetus(e.g. hamsters) and Cavia (e.g., guinea pigs) In general mammals with anormal full grown adult body weight of less than 1 kg which are easy tobreed and maintain can be used.

PrP Gene

[0120] The genetic material which makes up the PrP gene is known for anumber of different species of animals (see Gabriel et al., Proc. Natl.Acad. Sci. USA 89:9097-9101 (1992). Further, there is considerablehomology between the PrP genes in different mammals. For example, seethe amino acid sequence of mouse PrP compared to human, cow and sheepPrP in FIGS. 2, 3 and 4 wherein only the differences are shown. Althoughthere is considerable genetic homology with respect to PrP genes, thedifferences are significant in some instances. More specifically, due tosmall differences in the protein encoded by the PrP gene of differentmammals, a prion which will infect one mammal (e.g. a human) will notnormally infect a different mammal (e.g. a mouse). Due to this “speciesbarrier”, it is not generally possible to use normal animals, (i.e.,animal which have not had their genetic material related to PrP proteinsmanipulated) such as mice to determine whether a particular samplecontains prions which would normally infect a different species ofanimal such as a human. The present invention solves this problem byproviding antibodies which bind to native PrP^(Sc) proteins of anyspecies of animal for which the antibody is designed.

Pathogenic Mutations and Polymorphisms

[0121] There are a number of known pathogenic mutations in the human PrPgene. Further, there are known polymorphisms in the human, sheep andbovine PrP genes The following is a list of such mutations andpolymorphisms: Pathogenic human Human Sheep Bovine mutationsPolymorphisms Polymorphisms Polymorphisms 2 octarepeat Codon 129 Codon171 5 or 6 insert Met/Val Arg/Glu octarepeats 4 octarepeat Codon 219Codon 136 insert Glu/Lys Ala/Val 5 octarepeat insert 6 octarepeat insert7 octarepeat insert 8 octarepeat insert 9 octarepeat insert Codon 102Pro-Leu Codon 105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178Asp-Asn Codon 180 Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210Val-Ile Codon 217 Asn-Arg Codon 232 Met-Ala

[0122] The DNA sequence of the human, sheep and cow PrP genes have beendetermined allowing, in each case, the prediction of the complete aminoacid sequence of their respective PrP proteins. The normal amino acidsequence which occurs in the vast majority of individuals is referred toas the wild-type PrP sequence. This wild-type sequence is subject tocertain characteristic polymorphic variations. In the case of human PrP,two polymorphic amino acids occur at residues 129 (Met/Val) and 219(Glu/Lys). Sheep PrP has two amino acid polymorphisms at residues 171and 136, while bovine PrP has either five or six repeats of an eightamino acid motif sequence in the amino terminal region of the matureprion protein. While none of these polymorphisms are of themselvespathogenic, they appear to influence prion diseases. Distinct-from thesenormal variations of the wild-type PrP proteins, certain mutations ofthe human PrP gene which alter either specific amino acid residues ofPrP or the number of octarepeats have been identified which segregatewith inherited human prion diseases.

[0123] In order to provide further meaning to the above chartdemonstrating the mutations and polymorphisms, one can refer to thepublished sequences of PrP genes. For example, a chicken, bovine, sheep,rat and mouse PrP gene are disclosed and published within Gabriel etal., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992). The sequence forthe Syrian hamster is published in Basler et al., Cell 46:417-428(1986). The PrP gene of sheep is published by Goldmann et al., Proc.Natl. Acad. Sci. USA 87:2476-2480 (1990). The PrP gene sequence forbovine is published in Goldmann et al., J. Gen. Virol. 72:201-204(1991). The sequence for chicken PrP gene is published in Harris et al.,Proc. Natl. Acad. Sci. USA 88:7664-7668 (1991). The PrP gene sequencefor mink is published in Kretzschmar et al., J. Gen. Virol. 73:2757-2761(1992). The human PrP gene sequence is published in Kretzschmar et al.,DNA 5:315-324 (1986). The PrP gene sequence for mouse is published inLocht et al., Proc. Natl. Acad. Sci. USA 83:6372-6376 (1986). The PrPgene sequence for sheep is published in Westaway et al., Genes Dev8:959-969 (1994). These publications are all incorporated herein byreference to disclose and describe the PrP gene and PrP amino acidsequences.

“Strains” of Human Prions

[0124] Studies in rodents have shown that prion strains producedifferent patterns of PrP^(Sc) accumulation [Hecker et al., Genes &Development 6:1213-1228 (1992); DeArmond et al., Proc. Natl. Acad. Sci.USA 90:6449-6453 (1993)]; which can be dramatically changed by thesequence of PrP^(Sc) [Carlson et al., Proc. Natl. Acad. Sci. USA inpress (1994)]. The molecular basis of prion diversity has for many yearsbeen attributed to a scrapie specific nucleic acid [Bruce et al., J.Gen. Virol. 68:79-89 (1987)] but none has been found [Meyer et al., J.Gen. Virol. 72:37-49 (1991); Kellings et al., J. Gen. Virol.73:1025-1029 (1992)]. Other hypotheses to explain prion strains includevariations in PrP Asn-linked sugar chains [Hecker et al., Genes &Development 6:1213-1228 (1992)] and multiple conformers of PrP^(Sc)[Prusiner, S. B., Science 252:1515-1522 (1991)]. The patterns ofPrP^(Sc) in Tg(MHu2M) mice were remarkably similar for the three inoculafrom humans dying of CJD.

[0125] The patterns of PrP^(Sc) accumulation in the brains of inoculatedTg(MHu2M) mice were markedly different for RML prions and Hu prions.However, RML prion inocula containing MoPrP^(Sc) stimulated theformation of more MoPrP^(Sc) while Hu prion inocula containingHuPrP^(CJD) triggered production of MHu2MPrP^(Sc). The distribution ofneuropathological changes characterized by neuronal vacuolation andastrocytic gliosis is similar to the patterns of PrP^(Sc) accumulationin the brains of Tg(MHu2M) mice inoculated with RML prions or Hu prions.

Standardized Prion Preparation

[0126] Standardized prion preparations may be produced in order to testassays of the invention and thereby improve the reliability of theassay. Although the preparation can be obtained from any animal it ispreferably obtained from a host animal which has brain materialcontaining prions of a test animal. For example, a transgenic mousecontaining a human prion protein gene can produce human prions and thebrain of such a mouse can be used to create a standardized human prionpreparation. Further, in that the preparation is to be a “standard” itis preferably obtained from a battery (e.g., 100; 1,000, or moreanimals) of substantial identical animals. For example, 100 mice allcontaining a very high copy number of human PrP genes (all polymorphismsand mutations) would spontaneously develop disease and the brain tissuefrom each could be combined to make a useful standardized prionpreparation.

[0127] Standardized prion preparations can be produced using any ofmodified host mammals of the type described above. For example,standardized prion preparations could be produced using mice, rats,hamsters, or guinea pigs which are genetically modified so that they aresusceptible to infection with prions which prions would generally onlyinfect genetically diverse species such as a human, cow, sheep or horseand which modified host mammals will develop clinical signs of CNSdysfunction within a period of time of 350 days or less afterinoculation with prions. The most preferred host mammal is a mouse inpart because they are inexpensive to use and because a greater amount ofexperience has been obtained with respect to production of transgenicmice than with respect to the production of other types of host animals.Details regarding making standardized prion preparation are described inU.S. Patent application entitled “Method of Detecting Prions in a Sampleand Transgenic Animal Used For Same” filed Aug. 31, 1995, Ser. No.08/521,992 and U.S. Patent application entitled “Detecting Prions In ASample And Prion Preparation And Transgenic Animal Used For Same”,Attorney Docket No 06510/056001, filed Jul. 30, 1996, both of whichapplications are incorporated herein by reference.

[0128] Once an appropriate type of host is chosen, such as a mouse, thenext step is to choose the appropriate type of genetic manipulation tobe utilized to produce a standardized prion formulation. For example,the mice may be mice which are genetically modified by the insertion ofa chimeric gene of the invention. Within this group the mice might bemodified by including high copy numbers of the chimeric gene and/or bythe inclusion of multiple promoters in order to increase the level ofexpression of the chimeric gene. Alternatively, hybrid mice of theinvention could be used wherein mice which have the endogenous PrP geneablated are crossed with mice which have a human PrP gene inserted intotheir genome. There are, of course, various subcategories of such hybridmice. For example, the human PrP gene may be inserted in a high copynumber an/or used with multiple promoters to enhance expression. In yetanother alternative the mice could be produced by inserting multipledifferent PrP genes into the genome so as to create mice which aresusceptible to infection with a variety of different prions, i.e., whichgenerally infect two or more types of test animals. For example, a mousecould be created which included a chimeric gene including part of thesequence of a human, a separate chimeric gene which included part of thesequence of a cow and still another chimeric gene which included part ofthe sequence of a sheep. If all three different types of chimeric geneswere inserted into the genome of the mouse the mouse would besusceptible to infection with prions which generally only infect ahuman, cow and sheep.

[0129] After choosing the appropriate mammal (e.g., a mouse) and theappropriate mode of genetic modification (e.g., inserting a chimeric PrPgene) the next step is to produce a large number of such mammals whichare substantially identical in terms of genetic material related toprions. More specifically, each of the mice produced will include anidentical chimeric gene present in the genome in substantially the samecopy number. The mice should be sufficiently identical genetically interms of genetic material related to prions that 95% or more of the micewill develop clinical signs of CNS dysfunction within 350 days or lessafter inoculation and all of the mice will develop such CNS dysfunctionat approximately the same time e.g., within ±30 days of each other.

[0130] Once a large group e.g., 50 or more, more preferably 100 or more,still more preferably 500 or more of such mice are produced. The nextstep is to inoculate the mice with prions which generally only infect agenetically diverse mammal e.g., prions from a human, sheep, cow orhorse. The amounts given to different groups of mammals could be varied.After inoculating the mammals with the prions the mammals are observeduntil the mammals exhibit symptoms of prion infection e.g., clinicalsigns of CNS dysfunction. After exhibiting the symptoms of prioninfection the brain or at least a portion of the brain tissue of each ofthe mammals is extracted. The extracted brain tissue is homogenizedwhich provides the standardized prion preparation.

[0131] As an alternative to inoculating the group of transgenic micewith prions from a genetically diverse animal it is possible to producemice which spontaneously develop prion related diseases. This can bedone, for example, by including extremely high copy numbers of a humanPrP gene into a mouse genome. When the copy number is raised to, forexample, 100 or more copies, the mouse will spontaneously developclinical signs of CNS dysfunction and have, within its brain tissue,prions which are capable of infecting humans. The brains of theseanimals or portions of the brain tissue of these animals can beextracted and homogenized to produce a standardized prion preparation.

[0132] The standardized prion preparations can be used directly or canbe diluted and tittered in a manner so as to provide for a variety ofdifferent positive controls. More specifically, various known amounts ofsuch standardized preparation can be used to inoculate a first set oftransgenic control mice. A second set of substantially identical miceare inoculated with a material to be tested i.e., a material which maycontain prions. A third group of substantially identical mice are notinjected with any material. The three groups are then observed. Thethird group, should, of course not become ill in that the mice are notinjected with any material. If such mice do become ill the assay is notaccurate probably due to the result of producing mice whichspontaneously develop disease. If the first group, injected with astandardized preparation, do not become ill the assay is also inaccurateprobably because the mice have not been correctly created so as tobecome ill when inoculated with prions which generally only infect agenetically diverse mammal. However, if the first group does become illand the third group does not become ill the assay can be presumed to beaccurate. Thus, if the second group does not become ill the testmaterial does not contain prions and if the second group does become illthe test material does contain prions.

[0133] By using standardized prion preparations of the invention it ispossible to create extremely dilute compositions containing the prions.For example, a composition containing one part per million or less oreven one part per billion or less can be created. Such a composition canbe used to test the sensitivity of the antibodies, assays and methods ofthe invention in detecting the presence of prions.

[0134] Prion preparations are desirable in that they will include aconstant amount of prions and are extracted from an isogeneicbackground. Accordingly, contaminates in the preparations will beconstant and controllable. Standardized prion preparations will beuseful in the carrying out of bioassays in order to determine thepresence, if any, of prions in various pharmaceuticals, whole blood,blood fractions, foods, cosmetics, organs and in particular any materialwhich is derived from an animal (living or dead) such as organs, bloodand products thereof derived from living or dead humans. Thus,standardized prion preparations will be valuable in validatingpurification protocols where preparations are spiked and reductions inteeter measured for a particular process.

Useful Applications

[0135] As indicated above and described further below in detailedexamples it is possible to use the methodology of the invention tocreate a wide range of different antibodies. i.e., antibodies havingdifferent specific features. For example, antibodies can be createdwhich bind only to a prion protein naturally occurring within a singlespecies and not bind to a prion protein naturally occurring within otherspecies. Further, the antibody can be designed so as to bind only to aninfectious form of a prion protein (e.g., PrP^(Sc)) and not bind to anon-infectious form (e.g., PrP^(c)). A single antibody or a battery ofdifferent antibodies can then be used to create an assay device. Such anassay device can be prepared using conventional technology known tothose skilled in the art. The antibody can be purified and isolatedusing known techniques and bound to a support surface using knownprocedures. The resulting surface having antibody bound thereon can beused to assay a sample in vitro to determine if the sample contains oneor more types of antibodies. For example, antibodies which bind only tohuman PrP^(Sc) can be attached to the surface of a material and a samplecan be denatured via proteinase K. The denatured sample is brought intocontact with the antibodies bound to the surface of material. If nobinding occurs it can be deduced that the sample does not contain humanPrP^(Sc).

[0136] Antibodies of the invention are also characterized by theirability to neutralize prions. Specifically, when antibodies of theinvention are allowed to bind to prions the infectivity of the prion iseliminated. Accordingly, antibody compositions of the invention can beadded to any given product in order to neutralize any infectious prionprotein within the product. Thus, if a product is produced from anatural source which might contain infectious prion proteins theantibodies of the invention could be added as a precaution therebyeliminating any potential infection resulting from infectious prionproteins.

[0137] The antibodies of the invention can be used in connection withimmunoaffinity chromatography technology. More specifically, theantibodies can be placed on the surface of a material within achromatography column. Thereafter, a composition to be purified can bepassed through the column. If the sample to be purified includes anyprion protein which binds to the antibodies those prion proteins(PrP^(Sc)) will be removed from the sample and thereby purified.

[0138] Lastly, the antibodies of the invention can be used to treat amammal. The antibodies can be given prophylactically or be administeredto an individual already infected with infectious prion proteins suchinfection having been determined by the use of the assay describedabove. The exact amount of antibody to be administered will varydepending on a number of factors such as the age, sex, weight andcondition of the patient. Those skilled in the art can determine theprecise amount by administering antibodies in small amounts anddetermining the effect and thereafter adjusting the dosage. It issuggested that the dosage can vary from 0.01 mg/kg to about 300 mg/kg,preferably about 0.1 mg/kg to about 200 mg/kg, more preferably about 0.2mg/kg to about 20 mg/kg in one or more dose administrations daily, forone or several days. Preferred is administration of the antibody for 2to 5 or more consecutive days in order to avoid “rebound” of prioninfectivity occurring.

EXAMPLES

[0139] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the chimeric genes, transgenic mice and assays ofthe present invention, and are not intended to limit the scope of whatthe inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers used (e.g. amounts, temperature,etc.) but some experimental errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, molecularweight is weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Construction of Phage Display Antibody Libraries Expressing Antibodies(Fabs)

[0140] Construction of phage display libraries for expression ofantibodies, particularly the Fab portion of antibodies, is well known inthe art. Preferably, the phage display antibody libraries that expressantibodies are prepared according to the methods described in U.S. Pat.No. 5,223,409, issued Jun. 29, 1993 and U.S. patent application Ser. No.07/945,515, filed Sep. 16, 1992, incorporated herein by reference.Procedures of the general methodology can be adapted using the presentdisclosure to produce antibodies of the present invention.

Isolation of RNA Encoding Prion-specific Antibodies

[0141] In general, the phage display anti-PrP antibody libraries areprepared by first isolating a pool of RNA that contains RNA encodinganti-PrP antibodies. To accomplish this, an animal (e.g., a mouse, rat,or hamster) is immunized with prion of interest. However, normal animalsdo not produce antibodies to prions at detectable or satisfactorily highlevels. This problem is avoided by immunizing animals in which the (PrP)gene has been ablated on both alleles. Such mice are designatedPrnp^(0/0) and methods for making such mice are disclosed in Büeler,Nature (1992) and in Weismann Publication WO 93/10227, published May 27,1993. Inoculation of “null” animals with prions results in production ofIgG serum titers against the prion (Prusiner et al. PNAS 1993). In onepreferred embodiment, the animal selected for immunization is aPrnp^(0/0) mouse described by Bueler and Weismann.

[0142] Generally, the amount of prion necessary to elicit a serumantibody response in a “null” animal is from about 0.01 mg/kg to about500 mg/kg.

[0143] The prion protein is generally administered to the animal byinjection, preferably by intraperitoneal or intravenous injection, morepreferably by intraperitoneal injection. The animals are injected once,with at least 1 to 4 subsequent booster injections, preferably at least3 booster injections. After immunization, the reactivity of the animal'santisera with the prion can be tested using standard immunologicalassays, such as ELISA or Western blot, according to methods well knownin the art (see, for example, Harlow and Lane, 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). Animals having prion-binding antisera may be boosted withan additional injection of prion.

[0144] Serum antibody levels are predictive of antibody secretion, andtherefore of levels of specific mRNA in lymphocytes, particularly plasmacells. Detection of serum antibodies, particularly relatively highlevels of serum antibodies, is thus correlated to a high level oflymphocytes such as plasma cells producing mRNA encoding those serumantibodies. Thus, plasma cells isolated from the prion protein-immunizedmice will contain a high proportion of lymphocytes (e.g., plasma cells)producing prion-specific antibody, particularly when the plasma cellsare isolated from the mice within a short time period after the finalinjection boost (e.g., about 2 to 5 days, preferably 3 days).Immunization of the mice and the subsequent injection boosters thusserve to increase the total percentage of anti-PrP antibody-producingplasma cells present in the total population of the mouse's plasmacells. Moreover, because the anti-PrP antibodies are being produced ator near peak serum levels, then anti-PrP antibody-producing plasma cellsare producing anti-PrP antibodies, and thus mRNA encoding theseantibodies at or near peak levels.

[0145] The above correlation between serum levels of antigen-specificantibodies, the number of lymphocytes producing those antigen-specificantibodies, and the amount of total mRNA encoding the antigen-specificantibodies provides a means for isolating a pool of mRNA that isenriched for the mRNA encoding antigen-specific antibodies of interest.Lymphocytes, including plasma cells are isolated from spleen and/or bonemarrow from the prion-immunized animals according to methods well knownin the art (see, for example, Huse et al. Science 1989). Preferably thelymphocytes are isolated about 2 to 5 days, preferably about 3 daysafter the final immunization boost. The total RNA is then extracted fromthese cells. Methods for RNA isolation from mammalian cells are wellknown in the art (see, for example, Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.)

Production of cDNA Encoding Antibodies from Lymphocyte mRNA

[0146] cDNA is produced from the isolated RNA using reversetranscriptase according to methods well known in the art (see, forexample, Sambrook et al., supra), and cDNA encoding antibody heavychains or light chains is amplified using the polymerase chain reaction(PCR). The 3′ primers used to amplify heavy chain or lightchain-encoding cDNAs are based upon the known nucleotide sequencescommon to heavy chain or light chain antibodies of a specific antibodysubclass. For example, one set of primers based upon the constant regionof the IgG1 heavy chain-encoding gene can be used to amplify heavychains of the IgG1 subclass, while another set of primers based upon theconstant portion of the IgG1 light chain-encoding gene is used toamplify the light chains of the IgG1 subclass. The ′5 primers areconsensus sequences based upon examination of a large number of variablesequences in the data base. In this manner, DNA encoding all antibodiesof a specific antibody class or subclass are amplified regardless ofantigen-specificity of the antibodies encoded by the amplified DNA. Theentire gene encoding the heavy chain or the light chain can beamplified. Alternatively, only a portion of the heavy or light chainencoding gene may be amplified, with the proviso that the product of PCRamplification encodes a heavy or light chain gene product that canassociate with its corresponding heavy or light chain and function inantigen binding i.e., bind selectively to a prion protein. Preferably,the phage display product is a Fab or Fv antibody fragment.

[0147] The antibody encoding cDNA selected for amplification may encodeany isotope and preferably encode a subclass of IgG. Exemplary mouse IgGsubclasses include IgG1, IgG2a, IgG2b, and IgG3. The selection of thespecific antibody subclass-encoding cDNA for amplification will varyaccording to a variety of factors, including, for example, the animal'sserum antibody response to the antigen. Preferably, the antibodysubclass-encoding cDNA selected for PCR amplification is that antibodysubclass for which the animal produced the highest titer of antibody.For example, if the titers of serum IgG1 are higher than any othersubclass of IgG detected in the serum antibody response, then cDNAencoding IgG1 is amplified from the cDNA pool.

[0148] Preferably, the heavy and light chains are amplified from theplasma cell cDNA to produce two separate amplified cDNA pools: 1) a cDNApool containing heavy chain cDNA amplimer products, where the heavychain is of a specific antibody subclass; and 2) a cDNA pool containinglight chain cDNA amplimer products, where the light chain is of aspecific antibody subclass.

Antibodies From Transgenic Animals

[0149] In addition to obtaining genetic material which encodesantibodies by infecting an animal with an antigen and thereafterextracting cells (and their DNA) responsible for antibody production itis possible to obtain the genetic material by producing a transgenicanimal or by using the above described technology and transgenic animaltechnology in order to produce chimeric mouse/human or fully humanantibodies. The technology for producing a chimeric or wholly foreignimmunoglobins involves obtaining from cells of transgenic animals whichhave had inserted into their germ line a genetic material which encodesall or part of an immunoglobin which binds to the desired antigen.Wholly human antibodies can be produced from transgenic mice which havehad inserted into their genome genetic material which encodes humanantibodies. The technology for producing such antibodies from transgenicanimals is described within PCT Publication No. WO 90/04036, publishedApr. 19, 1990. Further, see Goodhartd, et al, Proc. Natl. Acad. Sci.U.S.A. Vol. 84, pages 4229-4233, June 1987 and Bucchine, et al, Nature,Vol. 326, pages 409-411, Mar. 26, 1987, all of which are incorporatedherein by reference to disclose and describe methods of producingantibodies from transgenic animals.

Vectors for Use with Phage Display Antibody Libraries

[0150] The heavy chain-encoding cDNAs and the light chain-encoding cDNAsare then each inserted into separate expression cassettes of anappropriate vector. Preferably the vector contains a nucleotide sequenceencoding and capable of expressing a fusion polypeptide containing, inthe direction of amino- to carboxy-terminus, 1) a prokaryotic secretionsignal domain, 2) an insertion site for DNA encoding a heterologouspolypeptide (e.g., either the heavy or light chain-encoding cDNA), andin the expression cassette for the heavy chain cDNA 3) a filamentousphage membrane anchor domain.

[0151] The vector includes prokaryotic or mammalian DNA expressioncontrol sequences for expressing the fusion polypeptide, preferablyprokaryotic control sequences. The DNA expression control sequences caninclude any expression signal for expressing a structural gene product,and can include 5′ and 3′ elements operatively linked to the expressioncassette for expression of the heterologous polypeptide. The 5′ controlsequence defines a promoter for initiating transcription, and a ribosomebinding site operatively linked at the 5′ terminus of the upstreamtranslatable sequence. The vector additionally includes an origin ofreplication for maintenance and replication in a prokaryotic cell,preferably a gram negative cell such as E. coli. The vector can alsoinclude genes whose expression confers a selective advantage, such asdrug resistance, to a prokaryotic or eukaryotic cell transformed withthe vector.

[0152] The filamentous phage membrane anchor is preferably a domain ofthe cpIII or cpVIII coat protein capable of associating with the matrixof a filamentous phage particle, thereby incorporating the fusionpolypeptide onto the phage surface. The secretion signal is a leaderpeptide domain of a protein that targets the protein to the periplasmicmembrane of gram negative bacteria. Such leader sequences for gramnegative bacteria (such as E. coli) are well known in the art (see, forexample, Oliver, In Neidhard, F. C. (ed.), Escherichia coli andSalmonella typhimurium, American Society for Microbiology, Washington,D.C., 1:56-69, 1987).

Filamentous Phage Membrane Anchors for Use in the Phage Display Vector

[0153] Preferred membrane anchors for the vector are obtainable fromfilamentous phage M13, f1, fd, and equivalent filamentous phage.Preferred membrane anchor domains are found in the coat proteins encodedby gene III and gene VIII. The membrane anchor domain of a filamentousphage coat protein is a portion of the carboxy terminal region of thecoat protein and includes a region of hydrophobic amino acid residuesfor spanning a lipid bilayer membrane, and a region of charged aminoacid residues normally found at the cytoplasmic face of the membrane andextending away from the membrane. In the page f1, gene VIII coatprotein's membrane spanning region comprises the carboxy-terminal 11residues from 41 to 52 (Ohkawa et al., J. Biol. Chem., 256: 9951-9958,1981). An exemplary membrane anchor would consist of residues 26 to 40to cpVIII. Thus, the amino acid residue sequence of a preferred membraneanchor domain is derived from the M13 filamentous phage gene VIII coatprotein (also designated cpVIII or CP 8). Gene VIII coat protein ispresent on a mature filamentous phage over the majority of the phageparticle with typically about 2500 to 3000 copies of the coat protein.

[0154] The amino acid residue sequence of another preferred membraneanchor domain is derived from the M13 filamentous phage gene III coatprotein (also designate cpIII). Gene III coat protein is present on amature filamentous phage at one end of the phage particle with typicallyabout 4 to 6 copies of the coat protein. Detailed descriptions of thestructure of filamentous phage particles, their coat proteins, andparticles assembly are found in the reviews by Rached et al.,(Microbiol. Rev., 50:401-427, 1986) and Model et al. (In: TheBacteriophages: Vol. 2, R. Calendar, ed., Plenum Publishing Co., pgs.375-456, 1988).

[0155] Preferably, the filamentous phage membrane anchor-encoding DNA isinserted 3′ of the cDNA insert in the library vector such that the phagemembrane anchor-encoding DNA can be easily excised and the vectorrelegated without disrupting the rest of the expression cassettes of thevector. Removal of the phage membrane anchor-encoding DNA from thevector, and expression of this vector in an appropriate host cell,results in the production of soluble antibody. (Fab) fragments. Thesoluble Fab fragments retain the antigenicity of the phage-bound Fab,and thus can be used in assays and therapies in the manner that whole(non-fragmented) antibodies are used.

[0156] The vector for use with the present invention must be capable ofexpressing a heterodimeric receptor (such as an antibody or antibodyFab). That is, the vector must be capable of independently containingand expressing two separate cDNA inserts (e.g., the heavy chain cDNA andthe light chain cDNA). Each expression cassette can include the elementsdescribed above, except that the filamentous phage anchormembrane-encoding DNA is present only in the expression cassette for theheavy chain cDNA. Thus, when the antibody or Fab is expressed on thesurface of the phage, only the heavy chain polypeptide is anchored tothe phage surface. The light chain is not directly bound to the phagesurface, but is indirectly bound to the phage via its association withthe free portion of the heavy chain polypeptide (i.e., the portion ofthe heavy chain that is not bound to the phage surface).

[0157] Preferably, the vector contains a sequence of nucleotides thatallow for directional ligation, i.e., a polylinker. The polylinker is aregion of the DNA expression vector that operatively links the upstreamand downstream translatable DNA sequence for replication and transport,and provides a site or means for directional ligation of a DNA sequenceinto the vector. Typically, a directional polylinker is a sequence ofnucleotides that defines two or more restriction endonucleaserecognition sequence, or restriction sites. Upon restriction enzymecleavage, the two sites yield cohesive termini to which a translatableDNA sequence can be ligated to the DNA expression vector. Preferably,the two cohesive termini are non-complementary and thereby permitdirectional insertion of the cDNA into the cassette. Polylinkers canprovide one or multiple directional cloning sites, and may or may not betranslated during expression of the inserted cDNA.

[0158] Preferably, the expression vector is capable of manipulating inthe form of a filamentous phage particle. Such DNA expression vectorsadditionally contain a nucleotide sequence that defines a filamentousphage origin of replication such that the vector, upon presentation ofthe appropriate genetic complement, can replicate as a filamentous phagein single stranded replicative form, and can be packaged intofilamentous phage particles. This feature provides the ability of theDNA expression vector to be packaged into phage particles for subsequentisolation of individual phage particles (e.g., by infection of andreplication in isolated bacterial colonies).

[0159] A filamentous phage origin of replication is a region of thephage genome that defines sites for initiation of replication,termination of replication, and packaging of the replicative formproduced by replications (see, for example, Rasched et al. Microbiol.Rev., 50:401-427, 1986; Horiuchi, J. Mol. Biol., 188:215-223, 1986). Apreferred filamentous phage origin of replication for use in the presentinvention is an M13, f1, or fd phage origin of replication (Short etal., Nucl. Acids Res., 16:7583-7600, 1988). Preferred DNA expressionvectors are the expression vectors pCOMB8, pCKAB8, pCOMB2-8, pCOMB3,pCKAB3, pCOMB2-3, pCOMB2-3′ and pCOMB3H.

[0160] The pComb3H vector is a modified form of pComb3 in which (i)heavy and light chains are expressed from a single Lac promoter asopposed to individual promoters and (ii) heavy and light chains have twodifferent leader sequences (pg1B and ompA) as opposed to the same leadersequence (pHB) Reference for pComb3H Wang, et al (1995) J. Mol. Biol.,Inpress. The principles of pComb3H are basically the same as for pComb3.

Production of the Phase Display Antibody Library

[0161] After the heavy chain and light chain cDNAs are cloned into theexpression vector, the entire library is packaged using an appropriatefilamentous phage. The phage are then used to infect a phage-susceptiblebacterial culture (such as a strain of E. coli), the phage allowed toreplicate and lyse the cells, and the lysate isolated from the bacterialcell debris. The phage lysate contains the filamentous phage expressingon its surface the cloned heavy and light chains isolated from theimmunized animal. In general, the heavy and light chains are present onthe phage surface as Fab antibody fragments, with the heavy chain of theFab being anchored to the phage surface via the filamentous phagemembrane anchor portion of the fusion polypeptide. The light chain isassociated with the heavy chain so as to form an antigen binding site.Method of producing chimeric antibodies are described within U.S. Pat.No. 4,816,567, issued Mar. 28, 1989 to Cabilly, et al which isincorporated herein by reference to disclose and describe suchprocedures. Further, See Bobrzecka, et al, Immunology Letters, 2, pages151-155 (1980) and Konieczny, et al, Haematologia 14 (1), pages 85-91(1981) also incorporated herein by reference.

Selection of Prion-antigen Specific Fabs from the Phage Display AntibodyLibrary

[0162] Phage expressing an antibody or Fab that specifically binds aprion antigen can be isolated using any of a variety of protocols foridentification and isolation of monoclonal and/or polyclonal antibodies.Such methods include, immunoaffinity purification (e.g., binding of thephage to a columna having bound antigen) and antibody panning methods(e.g., repeated rounds of phage binding to antigen bound to a solidsupport for selection of phage of high binding affinity to the antigen).Preferably, the phage is selected by panning using techniques that arewell known in the art.

[0163] After identification and isolation of phage expressing anti-PrPantibodies, the phage can be used to infect a bacterial culture, andsingle phage isolates identified. Each separate phage isolate can beagain screened using one or more of the methods described above. Inorder to further confirm the affinity of the phage for the antigen,and/or to determine the relative affinities of the phage for theantigen, the DNA encoding the antibodies or Fabs can be isolated fromthe phage, and the nucleotide sequence of the heavy and light chainscontained in the vector determined using methods well known in the art(see, for example, Sambrook et al., supra).

Isolation of Soluble Fabs from Phage Selected from the Phage DisplayAntibody Library

[0164] Soluble antibodies or Fabs can be produced from a modifieddisplay the same dicistronic vector by excising the DNA encoding thefilamentous phage anchor membrane that is associated with the expressioncassette for the heavy chain of the antibody. Preferably, the DNAencoding the anchor membrane is flanked by convenient restriction sitesthat allow excision of the anchor membrane sequence without disruptionof the remainder of the heavy chain expression cassette or disruption ofany other portion of the expression vector. The modified vector withoutthe anchor membrane sequence then allows for production of soluble heavychain as well as soluble light chain following packaging and infectionof bacterial cells with the modified vector.

[0165] Alternatively, where the vector contains the appropriatemammalian expression sequences the modified vector can be used totransform a eukaryotic cell (e.g., a mammalian or yeast cell, preferablya mammalian cell (e.g., Chinese hamster ovary (CHO) cells)) forexpression of the Fab. Where the modified vector does not provide foreukaryotic expression, preferably the vector allows for excision of boththe heavy and light chain expression cassettes as a single DNA fragmentsfor subcloning into an appropriate vector. Numerous vectors forexpression of proteins in prokaryotic and/or eukaryotic cells arecommercially available and/or well known in the art (see, for exampleSambrook et al., supra).

Commercial Assay

[0166] Examples 14-18 below and specifically Example 17 show theisolation of an antibody which specifically binds to PrP^(Sc) withoutany denaturation. A sample containing PrP proteins (i.e., PrP^(c) andPrP^(Sc)) can be subjected to denaturation by the use of protease K (PK)digestion. The use of such will digest PrP^(c) but not PrP^(Sc). Thus,after carrying out the digestion the sample is contacted with theantibody (e.g., R2) as per Example 17 under suitable binding conditions.Preferably, the antibody is bound to a substrate and can be positionedsuch that the sample can be easily contacted with the substrate materialhaving the antibody bound thereon. If material binds to the antibodieson the substrate the presence of infectious PrP^(Sc) is confirmed.

[0167] In commercial embodiments of the invention it may be desirable touse antibodies of the invention in a sandwich type assay. Moreparticularly, the antibody of the invention may be bound to a substratesupport surface. The sample to be tested is contacted with the supportsurface under conditions which allow for binding. Thereafter, unreactedsites are blocked and the surface is contacted with a generalizedantibody which will bind to any protein thereon. The generalizedantibody is linked to a detectable label. The generalized antibody withdetectable label is allowed to bind to any PrP^(Sc) bound to theantibodies on the support surface. If binding occurs the label can bemade to become detectable such as by generating a color therebyindicating the presence of the label which indirectly indicates thepresence of PrP^(Sc) within the sample. The assay can detect prions(PrP^(Sc)) present in an amount of 1 part per million or less, even onepart per billion or less. The PrP^(Sc) may be present in a sourceselected from the group consisting of (a) a pharmaceutical formulationcontaining a therapeutically active component extracted from an animalsource, (b) a component extracted from a human source, (c) an organ,tissue, body fluid or cells extracted from a human source, (d) aformulation selected form the group consisting of injectables, orals,creams, suppositories, and intrapulmonary delivery formulations, (e) acosmetic, and (f) a pharmaceutically active compound extracted from amammalian cell culture. Such source materials can also be treated toremove or neutralize PrP^(Sc) protein by adding an antibody of theinvention. The invention also includes a method of treating, comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of an antibody which selectively binds PrP^(Sc) protein whichantibody is characterized by its ability to neutralize PrP^(Sc) proteininfectivity.

Generalized Procedure

[0168] Antibodies of the invention could be obtained by a variety oftechniques. However, the general procedure involves synthesizing alibrary of proteins (i.e., antibodies or portions thereof) on thesurface of phage. The library is then brought into contact with acomposition which includes PrP proteins and in particular is a naturallyoccurring composition which includes PrP^(Sc). The phage which bind toPrP protein are then isolated and the antibody or portion thereof whichbinds the PrP protein is isolated. It is desirable to determine thesequence of the genetic material encoding the antibody or portionthereof. Further, the sequence can be amplified and inserted, by itself,or with other genetic material into an appropriate vector and cell linefor the production of other antibodies. For example, a sequence encodinga variable region which binds PrP^(Sc) can be fused with a sequencewhich encodes a human constant region of an antibody producing aconstant/variable construct. This construct can be amplified andinserted within a suitable vector which can be inserted within asuitable cell line for the production of humanized antibodies.Procedures such as this are described within U.S. Pat. No. 4,816,567,issued Mar. 28, 1989 to Cabilly, et al which is incorporated herein byreference to disclose and describe such procedures. Further, SeeBobrzecka, et al, Immunology Letters, 2, pages 151-155 (1980) andKonieczny, et al, Haematologia 14 (1), pages 85-91 (1981) alsoincorporated herein by reference.

[0169] When the genetic material encoding an antibody or portion thereofwhich binds a PrP protein is isolated it is possible to use that geneticmaterial to produce other antibodies or portions thereof which have agreater affinity for binding PrP proteins. This is done by site directedmutagenesis technology or by random mutagenesis and selection.Specifically, individual codons or groups of codons within the sequenceare removed or replaced with codons which encode different amino acids.Large numbers of different sequences can be generated, amplified andused to express variations of the antibody or portions thereof on thesurface of additional phage. These phage can then be used to test forthe binding affinity of the antibody to PrP proteins.

[0170] The phage library can be created in a variety of different ways.In accordance with one procedure a host animal such as a mouse or rat isimmunized with PrP protein and preferably immunized with PrP^(Sc). Theimmunization may be carried out along with an adjuvant for the formationof larger amounts and types of antibodies. After allowing for sufficienttime for the generation of antibodies, cells responsible for antibodyproduction are extracted from the inoculated host mammal. RNA isisolated from the extracted cells and subjected to reverse transcriptionin order to produce a cDNA library. The extracted cDNA is amplified bythe use of primers and inserted into an appropriate phage displayvector. The vector allows the expression of antibodies or portionsthereof on the phage surface. It is also possible to subject the cDNA tosite directed mutagenesis prior to insertion into the display vector.Specifically, codons are removed or replaced with codons expressingdifferent amino acids in order to create a larger library (i.e., alibrary of many variants) which is then expressed on the surface of thephage. Thereafter, as described above, the phage are brought intocontact with the sample and phage which bind to PrP protein areisolated.

EXAMPLES

[0171] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the recombinant anti-PrP antibodies and assays ofthe present invention, and are not intended to limit the scope of whatthe inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers used (e.g. amounts, temperature,etc.) but some experimental errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, molecularweight is weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Purification of MoPrP 27-30

[0172] Purified MoPrP 27-30 rods were prepared from the brains ofclinically ill CD-1 mice inoculated with RML prions (Chandler scrapieisolate (Chandler R. L. 1961 Lancet, 1378-1379)). Prion rods wererecovered from sucrose gradient fractions as previously described(Prusiner, McKinley 1983 Cell). Briefly, the fractions containing prionrods, which sediment in 48-60% (wt/vol) sucrose, were diluted 2:1 indistilled water and centrifuged at 100,000×g for 6 h at 4° C. The pelletwas resuspended in water, centrifuged again, and the rods resuspended at1 mg/ml in Ca/Mg-free phosphate buffered saline (PBS) containing 0.2%Sarcosyl. PrP 27-30 was the major protein as determined by SDS-PAGE andsilver staining analysis. Protein quantization was performed bybicinchonic acid dye binding, with a known amount of bovine serumalbumin as the protein concentration standard.

Example 2 Immunization of Prnp^(0/0) Mice

[0173] Prnp^(0/0) mice, in which both alleles of the PrP gene (Prnp) isablated, were immunized with the purified MoPrP 27-30 rods, which wereisolated as described in Example 1. Prnp^(0/0) mice and methods formaking this strain are well known in the art (Bueler, et al. 1992).Prnp^(0/0) mice, which are indistinguishable from normal mice in theirdevelopment and behavior, are resistant to scrapie followingintracerebral inoculation of infectious MoPrP^(Sc) (Bueler, et al. 1993Cell; Prusiner et al. PNAS 1993), and will develop IgG serum titersagainst Mo-, SHa, and human PrP following immunization with relativelysmall quantities of purified SHaPrP 27-30 in adjuvant (Prusiner et al.PNAS 1993).

[0174] Three (3) six week old Prnp^(0/0) mice were immunized byintraperitoneal injection of 100 μg of MoPrP 27-30 rods fully emulsifiedin complete Freund's adjuvant. Subsequently mice were boosted 2 times at2-week intervals with incomplete Freund's adjuvant containing in thefirst instance 100 μg, then 50 μg of rods. Four days after the secondboost, the reactivity of each mouse's serum against prion proteins wasanalyzed as described below in Example 3. Those mice having anti-PrPreactive antisera received a third injection boost of 50 μg prion rodsin incomplete Freund's adjuvant 14 days after the second boost.

Example 3 Serum Reactivity of Prnp^(0/0) Mice Immunized with MoPrP 27-30

[0175] A primary prognostic indicator for success in isolating aspecific antibody from combinatorial libraries is serum antibodyreactivity with the antigen(s) to be studied (Burton and Barbas, Adv.Immunol. 1994). Serum antibody levels are predictive of antibodysecretion and therefore predictive of the levels of specific mRNA inplasma cells. It is this latter factor that ultimately dictates thecomposition of the antibody-encoding cDNA library.

[0176] Four days after the second boost, the Prnp^(0/0) mice immunizedwith MoPrP 27-30 as described in Example 2 were bled from the tail, andthe antisera stored at −20° C. for subsequent immunological analysis.The reactivity of the immunized mouse serum (IgG1, IgG2a, IgG2b and IgG3antibody subclasses) was measured against denatured and non-denaturedMo- and SHaPrP 27-30 in ELISA. ELISA wells were coated overnight at 4°C. with 50 μl of PrP rods at 40 μg/ml in 100 mM sodium bicarbonate pH8.6. Where denatured PrP rods were used as the antigen in the ELISA, 50μl of 6M guanidinium isothiocyanate was added to the well for 15 min atroom temperature, after which the wells were washed 6 times withCa/Mg-free PBS. All wells were then blocked with Ca/Mg-free PBScontaining 3% BSA. The antisera was serially diluted in PBS, andincubated with the wells for one hour at 37° C. Excess antisera wasremoved by washing 10 times with PBS 10.05% Tween 20 and bound antiseradetected using labeled goat anti-mouse antibody that specifically bindseither IgG1, IgG2a, IgG2b or IgG3 murine antibodies.

[0177] All 3 mice produced anti-PrP IgG antibodies. Serum reactivityfrom one of the mice, designated D7282, is illustrated in FIG. 5 asexemplary of the antibody responses of the immunized mice. The highestserum titers against Mo- and SHaPrP antigens were of the IgG1 and IgG2bsubclasses. In contrast, the IgG2a and IgG3 anti-PrP titers were closeto the background levels of reactivity seen for all IgG subclasses inthe serum of non-immunized Prnp^(0/0) mice. Antibody titers were greateragainst denatured rods than non-denatured rods. The similar serumreactivity against Mo- and SHa denatured rods is likely reflective ofthe high amino acid sequence homology between the two proteins. However,although there was considerable serum reactivity against non-denaturedMo- rods (approximately 40-50% of the level of that for denatured MoPrP27-30), reactivity with non-denatured SHa rods was at the level ofbackground.

Example 4 Isolation of mRNA Encoding Anti-PrP Antibodies andConstruction of Antibody Phage Display Libraries

[0178] Three days after the final injection boost, the D7282 mouse wassacrificed and RNA prepared from bone marrow and splenic tissues. TotalRNA from mouse spleen was prepared according to methods well known inthe art (Huse, et al Science 1989). RNA was prepared from bone marrowtissues by first removing the tibia and fibula from both rear legs ofthe mice. The bones were then cut through close to each end, and theircontents flushed out by injection of guanidinium isothiocyanate into thebone cavity using a 27 gauge needle. RNA preparation was then continuedas described for the mouse spleen.

[0179] The RNA preparations were then pooled, and cDNA generated fromthe mRNA using reverse transcriptase according to methods well known inthe art. Two cDNA libraries were independently constructed from theD7282 mouse mRNA: 1) an IgG1 library; and 2) a IgG2b library. For eachof these libraries, cDNAs encoding heavy chains and cDNA light chainswere separately amplified by PCR from separate fractions of the pooledcDNA. The oligonucleotide 5′ and 3′ primers employed for PCRamplification of DNA fragments encoding murine light (K) chains andheavy (α1 or α2b) chains of the IgG1 subclass wee those used by Huse, etal (Science 1989) and additional heavy chain primers as presented inTable 1 and heavy chain polymers which are presented in Table 1. Primersused for amplification of cDNAs encoding heavy chain fragments. TABLE 1Primer Nucleotide Sequence HEAVY CHAIN PRIMERS MVH 1b 5′-[CG]AG GTG CAGCTC GAG GAG TCA GGA CCT-3′ MVH 2b 5′-GAG GTC CAG CTC GAG CAG TCT GGACCT-3′ MVH 3b 5′-CAG GTC CAA CTC GAG CAG CCT GGG GTC-3′ MVH 4b 5′-GAGGTT GAG CTC GAG CAG TCT GGG GCAA-3′ MVH 5b 5′-GA[AG]GTG AAG CTC GAG GAGTCT GGA GGA-3′ MVH 6b 5′-GAG GTG AAG CTT CTC GAG TCT GGA GGT-3′ MVH 7b5′-GAA GTG AAG CTC GAG GAG TCT GGG GGA-3′ MVH 8b 5′-GAG GTT CAG CTC GAGGAG CAG TCT GGA GCT-3′ MVH 1a 5′-AGG T[CG] [CA] A[GA]C T[GT]C TCG AGTC[TA]GG-3′ MVH 2a 5′-AGG TCC AGC TGC TCG AGT CTG G-3′ MVH 3a 5′-AGG TCCAGC TGC TCG ACT CAG G-3′ MVH 4a 5′-AGG TCC AGC TTC TCG AGT CTG G-3′ MVH5a 5′-AGG TCC AGC TTC TCG AGT CAG G-3′ Primers used for theAmplification of Antibody Light Chain Fragments 5′ PRIMERS MVK 1 5′-CCAGTT CCG AGC TCG TTG TGA CTC AGG AAT CT-3′ MVK 2 5′-CCA GTT CCG AGC TCGTGG TGA CGC AGC CGC CC-3′ MVK 3 5′-CCA GTT CCG AGC TCG TGC TCA CCC AGTCTC CA-3′ MVX 4 5′-CCA GTT CCG AGC TCC AGA TGA CCC AGT CTC CA-3′ MVX 55′-CCA GAT GTG AGC TCG TGA CCC AGA CTC CA-3′ MVK 6 5′-CCA GAT GTG AGCTCG TCA TGA CCC AGT CTC CA-3′ MVK 7 5′-CCA GTT CCC AGC TCG TGA TGA CACAGT CTC CA-3′ 3′ PRIMERS MCK 1 5′-GCG CCG TCT AGA ATT AAC ACT CAT TCCTGT TGA A-3′ MVH 6a 5′-AGG TCC AAC TGC TCG AGT CTG G-3′ MVH 7a 5′-AGGTCC AAC TGC TCG AGT TCA G-3′ MVH 8a 5′-AGG TCC AAC TTC TCG AGT CTG G-3′3′ PRIMERS MIgGI 5′-AGG CTT ACT AGT ACA ATC CCT GGG CAC AAT-3′ MIgG2B5′-CTC CTT ACT AGT AGG ACA GGG GAT TGT-3′

[0180] PCR was performed using a Perkin Elmer 9600 with 35 rounds ofamplification; denaturation at 94° C. for 30 sec, hybridization at 52°C. for 60 sec and extension at 72° C. for 60 sec.

[0181] The resulting amplified cDNAs encoding heavy chains of the IgG1and IgG2b subclasses and light chains were cloned into the vectorpComb3. The preparation of Fab antibody libraries displayed on thesurface of a filamentous phage using the pComb3 vector have beendescribed (Williamson et al. PNAS, 1993; Barbas et al. PNAS 1991).Briefly, the IgG1 or IgG2b phage display library is constructed byinserting the amplified cDNA encoding IgG1 or IgG2b heavy chain and theamplified cDNA encoding light chain into the pComb3H vector such thateach vector contains a cDNA insert encoding a heavy chain fragment inone expression cassette of the vector, and a cDNA insert encoding alight chain fragment into the other expression cassette of the vector.The resulting IgG1 library contained approximately 9×10⁶ individualclones, while the resulting IgG2b library contained approximately 7×10⁶individual clones.

[0182] The ligated vectors were then packaged by the filamentous phageM13 using methods well known in the art (see, for example, Sambrook etal, supra). The packaged library is then used to infect a culture of E.coli, so as to amplify the number of phage particles. After bacterialcell lysis, the phage particles are isolated and used in the panningprocedure that follows. Aliquots of the phage library are stored forfuture amplification and use. Separate aliquots of the phage librariesare isolated and stored for future amplification and use.

Example 5 Screening of the Phage Display Antibody Library for Binding toPrP

[0183] Antigen binding phage were selected for binding to denaturedMoPrP 27-30 rods against PrP antigen bound to ELISA wells through apanning procedure described in (Burton, et al PNAS 1991, Barbas LernerMethods in Enzymol 1991). Briefly, ELISA wells were coated overnight at4° C. with 50 μl of MoPrP 27-30 rods at 40 μg/ml in 100 mM sodiumbicarbonate pH 8.6. The PrP rods were then denatured by incubation with50 μl of 6M guanidinium isothiocyanate for 15 min at room temperature,after which the wells were washed 6 times with Ca/Mg-free PBS. The wellswere then blocked with Ca/Mg-free PBS containing 3% BSA.

[0184] Aliquots of antibody phage were applied to separate PrP coatedELISA wells. A total of approximately 1×10¹⁰ antibody phage were addedper well in the panning experiment.

[0185] The phage were incubated with the well-bound MoPrP antigen for 2hrs at 37° C. Unbound phage were removed by washing 10 times with PBS0.5% TWEEN 20. Bound phage were then removed from the wells by acidelution, pooled, reamplified and subjected to a second round of panning.

[0186] The IgG1 library was selected through 5 rounds of panning. A40-fold amplification of PrP-specific antibody phage, as determined bythe number of phage eluted from PrP-coated ELISA wells, was measuredfrom the first to the fifth round.

Example 6 Soluble Fab Production from Selected Antibody-producing Phage

[0187] Soluble Fabs were produced from phage clones eluted from thefourth and fifth rounds of panning. DNA from the selected phage cloneswas isolated, and the phage coat protein III (the filamentous phagemembrane anchor) was removed from the pComb3H vector using theappropriate restriction enzymes. The DNA was self-ligated to yield avector capable of expressing soluble Fab (the procedure for productionof soluble Fabs is detailed in (Barbas et al. PNAS 1991)). The vectorswere then separately used to transform bacteria for expression of theFabs, and isolated transformants were selected.

[0188] Fab expression was induced in an overnight bacterial cultureusing isopropyl β-D-thiogalactopyranoside. The bacteria werecentrifuged, and the resulting bacterial pellet was either sonicated orfrozen and thawed three times to release Fab from the bacterialperiplasmic space. The bacterial Fab supernatants were then tested forreactivity against PrP in ELISA.

Example 7 ELISA Analysis of Anti-PrP Fabs Binding to PrP Antigens

[0189] The binding of soluble Fabs produced in Example 6 to denaturedand non-denatured PrP antigens as well as to synthetic PrP peptides wasdetermined using the ELISA assay described in Example 3. Synthetic PrPpeptides were produced using conventional peptide synthesis protocolswell known in the art.

[0190] Of the Fab clones taken from the fourth round of the panningagainst denatured MoPrP rods, less than 5% were reactive with denaturedPrP, while approximately 50% of the clones taken from the fifth round ofthe same panning recognized PrP antigens. In ELISA all of the reactiveclones from this panning were able to bind specifically to denatured Moand SHa rods, but not to non-denatured rods from either species. Inaddition, all the anti-PrP Fabs failed to recognize synthetic peptidesspanning residues 90-145 of Mo and SHa PrP, suggesting the antibodiesbind between residues 146 and 231 of the prion protein.

Example 8 Analysis of Selected Anti-PrP Antibody (Fab) Binding toPrion-infected and Uninfected Rodent Brain Tissue

[0191] The reactivity of the antibodies identified by panning of thephage display antibody library was tested by SDS/PAGE of prion-infectedrodent brain tissue and Western blot analysis using the selected Fabs.Protein from brain tissues of prion-infected and uninfected mice wasused as the antigen against which immunoreactivity was tested. Theantigen was prepared by disrupting rodent brain tissue in Ca/Mg-free PBSby passage 5 times through a 20 gauge needle, followed by passage 10times through a 22 gauge needle. The 10% (wt/vol) homogenate was thencentrifuged at 1600×g for 5 min at 4° C. Aliquots of the supernatantprotein were diluted to a final concentration of 1 mg/ml in Ca/Mg-freePBS containing 0.2% Sarcosyl. This dilution was mixed with an equalvolume of non-reducing 2×SDS/PAGE sample buffer and boiled for 5 min,before SDS/PAGE (Laemmli. U.K. (1970) Nature (London) 227, 680-685).Immunoblotting was performed as previously described (Pan et al, PNAS1993) with primary mouse IgG antiserum (Pierce) diluted 1:1000.

Example 9 Nucleic Acid Sequencing

[0192] The nucleotide and amino acid sequences of the variable domainsof the antibody light and heavy chains were determined for several ofthe PrP specific clones. Nucleic acid sequencing was performed with amodel 373A automated DNA sequencer (Applied Biosystems) using a Taqfluorescent dideoxynucleotide terminator cycle sequencing kit (AppliedBiosystems). Primers for the elucidation of antibody light-chainsequence were primers MoSeqKb [5′-CAC GAC TGA GGC ACC TCC-3′] and OmpSeq[5′-AAG ACA GCT ATC GCG ATT GCA G-3′] hybridizing to the (−)-strand andfor the heavy chain MOIgGGzSeq [5′-ATA GCC CTT GAC CAG GCA TCC CAG CGTCAC-3] binding to the (+)-strand and PelSeq [5′-ACC TAT TGC CTA CGG CAGCCG-3′] binding to the (−)-strand.

[0193] The deduced amino acid sequences for some of the phage clonesobtained in one panning against denatured PrP are provided in FIGS. 6and 7. FIG. 6 shows the amino acid sequences of selected (A) heavy chainand (B) light chain variable regions generated by panning an IgG1library from mouse D7282 against denatured MoPrP 27-30 rods. Thesequences are very similar but contain a number of heterogeneities whichare likely the result of somatic mutation following repeated exposure ofthe mouse to PrP antigen. All of the heavy chain sequences examined inthese clones contained very similar sequences. In particular, the heavychain complementarity determining region 3(HCDR3) was identical at thenucleotide level in all the Fab clones examined. Small differences wereobserved in the CDR1, CDR2, framework (FR) 3 and FR4 of the heavy chain.These differences are too numerous to be attributable to PCR orsequencing errors and have probably accrued during rounds of somaticmutation as the mouse was repeatedly boosted with antigen. The lightchain sequences were also very similar, but with localized heterogeneitythroughout the variable domain, again probably resultant of somaticmutation.

Example 10 Selection of Anti-prion Antibodies Following Masking ofEditopes With Existing Antibodies

[0194] Panning of the IgG1 library against denatured PrP produced aseries of related antibodies, presumably somatic variants of a clonedirected to a single epitope (Example 9). To access antibodies to otherepitopes, a prototype antibody from the above series was added todenatured PrP in ELISA wells prior to panning in the normal way. Themasking antibody was used in all subsequent panning steps. Using thisprocedure, antibodies were derived of different sequence which reactedwith denatured PrP in ELISA. These antibodies are likely directed todifferent epitopes on PrP. The masking procedure was carried out asdescribed in Ditzel, et al (1995) J. Immunol. Masking could also becarried out with molecules other than antibodies which interacted withPrP.

Example 11 Selection of Phage Particles Expressing Anti-PrP AntibodiesSpecific for PrP^(Sc)

[0195] A phage display antibody library similar to that described in theExamples above is subjected to panning experiments to identify phageclones that bind to PrP^(Sc), but not to PrP^(c). PrP^(Sc) antigen andPrP^(c) antigen are bound to separate wells of a microtiter dish asdescribed above for the ELISA assay. The phage display antibody libraryis first panned over the PrP^(c) ELISA wells. Unbound phage areretrieved from the wells and pooled. Phage that binds to the PrP^(c)antigen are removed from the wells and either discarded or pooled forlater analyses. The pooled unbound phage are then again added to PrP^(c)ELISA wells, with selection again being based upon lack of binding tothe PrP^(c). After several repeated selections on the PrP^(c) antigen,the phage are pooled and panned on the ELISA wells containing thePrP^(Sc) antigen. The panning is repeated for several rounds, with thephage that binds to the PrP^(Sc) antigen being the phage that isselected for further rounds of panning. After 5 to 10 rounds of panningon the PrP^(Sc) antigen, the phage are isolated one from another. Theability of the PrP^(Sc)-specific phage or isolated Fab to bind PrP^(c)antigen can be double-checked by ELISA with the PrP^(c) antigen. Theresulting selected phage are those that bind PrP^(Sc), but do not bindPrP^(c).

Example 12 Selection of Phage Particles Expressing Anti-PrP Antibodiesto Identify PrP^(Sc) regardless of Isoform

[0196] A phage display antibody library is prepared as described abovefrom lymphocyte RNA from a mouse immunized with several PrP^(Sc)isoforms, or from a pool of lymphocyte RNA from several mice immunizedwith different PrP^(Sc) isoforms. The phage are then panned with severaldifferent wells containing antigens from different isoforms of PrP^(Sc).The phage are panned over each PrP^(Sc) isoform with the selection beingfor phage that bind the isoform at each stage. The phage are panned fora total of about 5 to 10 rounds on each PrP^(Sc) isoform. The phage thatremain after all stages of panning against all the isoforms tested arethen isolated. The immunoreactivity of each selected phage or isolatedFab is tested by ELISA or Western blot or histochemistry against each ofthe various PrP^(Sc) isoforms, as well as for cross-reactivity withPrP^(c).

Example 13 Selection of Phage Particles Expressing Anti-PrP AntibodiesSpecific for Isoforms of PrP^(Sc)

[0197] A phage display antibody library prepared from lymphocyte RNA ofa mouse immunized with a specific PrP^(Sc) isoform is prepared accordingto the Examples above. The resulting phage are then selected for theirability to bind only one specific PrP^(Sc) isoform by panning. Thepanning uses several different wells containing antigens from differentisoforms of PrP^(Sc), including one set of wells containing antigensfrom the specific PrP^(Sc) isoform against which specific antibodies aredesired. The phage are first panned over the undesirable PrP^(Sc)isoforms, with the selection being for phage that do not bind theantigen. Panning continues for a total of about 5 to 10 rounds on eachof the PrP^(Sc) isoforms. The phage that did not bind the undesirablePrP^(Sc) isoforms are then panned for about 5 to 10 rounds against thedesirable PrP^(Sc) isoform, with selection for antigen binding. Thephage that remain after all rounds of panning are isolated. Theseselected phage are those that express antibodies with bindingspecificity for only the specific PrP^(Sc) isoform desired. Theimmunoreactivity of each selected phage or isolated Fab is tested byELISA or Western blot against each of the various PrP^(Sc) isoforms, aswell as for cross-reactivity with PrP^(c).

Example 14 Generation and Characterization of Serum Reactivity AgainstPrP^(Sc) In PrP^(c) Mice

[0198] Experimentation per the above Examples established that theprimary prognostic indicator for success in isolating a specificantibody from combinatorial libraries with the size range of 10⁷ pfu/mlis the serum reactivity with the antigen to be studied, and it is thisfactor which will ultimately dictate the composition of the library.Although Prnp^(0/0) mice elucidated a strong immune response uponimmunization with either mouse (Mo) or Syrian hamster (SHa) prion rodscomposed of PrP 27-30 proteins, the highest serum titers were seen inthe IgG1 and IgG2b subclasses. The IgG2a and IgG3 anti-PrP titers wereclose to the background levels of reactivity seen for all IgG subclassesin the serum of non-immunized mice. In an attempt to increase the immuneresponse and augment the immune repertoire against PrP^(Sc), Prnp^(0/0)(94% FVB) female mice were immunized with liposomes containing SHaPrP27-30. To further increase the immune response diversity, mice wereimmunized using both short and long term protocols. In contrast toimmunization with SHa prion rods immunization with liposomes containingSHaPrP 27-30 resulted in antiserum titer which includes all four IgGsubclasses.

Example 15 PrP-immunized Sera Reactivity Against Histoblots

[0199] To further investigate the properties of the IgG anti-SHaPrP27-30 found in the sera from mice immunized with liposomes containingSHaPrP 27-30, we tested the sera in situ with histoblotting techniques,in which cryostat sections of normal and scrapie infected SHa brain weretransferred onto nitrocellulose membranes. Although both sera showedsome nonspecific reactivity against proteinase K (PK)-treated normal SHabrain sections, only the sera from the long term immunized mice showedincreased reactivity against PK-treated SHa scrapie infected brainsections. This reactivity was also evident in sera dilution to {fraction(1/1000)} (results not shown). Both sera showed typical reactivityagainst SHa scrapie infected brain sections which were first PK-treatedand then exposed to 3M GdnSCN for 10 minutes. Sera from non-immunizedPrnp^(0/0) (94% FVB) female mice did not show any immune reactivityagainst normal scrapie infected SHa brain sections.

Staining of SHaPrP 27-30 and Denatured SHaPrP 27-30 in Histoblots ofScrapie Infected SHa Brain

[0200] Histoblots were treated with proteinase K to remove PrP^(c) fromthe brain of normal, uninoculated control SHa and SHa showing clinicalsigns of scrapie following inoculation with Sc237 prions. To denatureSHaPrP 27-30, histoblots were treated with 3M GdnSCN for 10 minutes.Blots were incubated overnight at 4° C. with sera diluted {fraction(1/200)} from the short and the long term immunized mice. The resultsdescribed here show clear positive reactivity of an antiserum withnon-denatured infectious prions i.e., native PrP^(Sc).

[0201]FIG. 8 shows eight different stained histoblots of scrapieinfected SHa brain. The histoblots were treated with proteinase K toremove PrP^(c) from the brain of normal, non-inoculated control SHa(A,C, E and G) and SHa showing clinical signs of scrapie followinginoculation with Sc 237 prions (B, D, F and H). To denature the SHaPrP27-30, the histoblots were treated with 3M GdnSCN for 10 minutes (C, D,G and H). The blots were incubated overnight at 4° C. with sera diluted{fraction (1/200)} from the short (A-D) and the long (E-H) termimmunized mice. The results clearly show the ability of the antibodiesof the invention to bind to native, non-denatured infectious prionsi.e., bind to native PrP^(Sc).

Example 16 Generation Of Monoclonal Antibodies From immunized Mice OfExample 14

[0202] Overall, eight phage Fab display libraries were constructed:IgG1k, IgG2ak, IgG2bk and IgG3k from mRNA extracted from the short andlong term immunized mice. To overcome difficulties with the isolation ofphage expressing anti-PrP Fab by panning against prion rods containingPrP 27-30, a panning system was used where libraries are panned againstbiotinylated SHa 27-30, dispersed into liposomes, and bound tostreptavidin-coated microtiter plates. After five rounds of panning, E.coli extracts from more than 50 clones reacted with biotinylated SHa27-30, SHa 27-30 rods and 90-231 recombinant SHa in ELISA. Since theseclones also react with recombinant rPrP corresponding to SHaPrP residues90-231, Melhorn, I., et al, High-level Expression and Characterizationof a Purified 142-residue Polypeptide of the Prion Protein. Biochemistry35, 5528-2237 (1996), all eight libraries were panned against thisantigen to successfully isolate more distinct clones from virtually allthe libraries. Upon DNA sequencing of the plasmid region coding for theIgG heavy chain, 30 Fabs were identified as distinct clones.

Example 17 Characterization of Monoclonal Antibodies

[0203] Initial ELISA with E. coli extracts from positive clonessuggested that the Fabs, in contrast to the monoclonal 3F4 antibody,Kascsak, R. J., et al, Mouse Polyclonal and Monoclonal Antibody toScrapie Associated Fibril Proteins, J. Virol; 61, 3688-3693 (1987), bindto PrP 27-30 in a native state, i.e., without a denaturation step. Tocharacterize quantitatively the novelty of these Fabs, we purified themand produced 3F4 Fab from the monoclonal 3F4 by enzymatic cleavage.Standard ELISA for the detection of SHaPrP was performed using differentconcentrations of the purified Fabs. In contrast to 3F4 which showedcharacteristic SHa PrP binding properties (basal binding to prion rodsand strong reactivity against SHaPrP 27-30 after treatment with 3Mnon-denaturant GdnSCN), the newly isolated Fabs reacted against prionrods without any denaturation step. The half-maximal binding tonon-denatured prion rods occurs at a Fab concentration of approximately0.5 pg/ml, indicating that the antibody has an apparent binding affinityof approximately 10⁸ moles/liter.

[0204]FIG. 9 is a graph showing the ELISA reactivity of purified Fabsagainst prion protein SHa 27-30. The antibody 3F4 and recombinantantibodies were examined at different concentrations for binding toELISA wells which were coated with: 0.2 μ/g of sucrose purifiedinfectious SHa prion rods. The results clearly show that all of therecombinant antibodies of the invention have substantially higherdegrees of binding to prions as compared to the antibody 3F4.

Protocol For ELISA Reactivity Of Purified Fabs Against Denatured PrionProtein SHa 27-30

[0205] Purified 3F4 Fab and recombinant Fabs were examined at differentconcentrations for binding to ELISA wells coated with 0.2 μg of sucrosepurified SHa prion rods either native or denatured in the ELISA wellwith 3M GdnSCN for 10 min.

[0206]FIG. 10 is a graph showing the results of ELISA reactivitypurified Fabs against denatured prion protein SHa 27-30. FIG. 10 isinteresting as compared to FIG. 9 in that the recombinant antibodies ofthe invention as per FIG. 9 show a higher degree of affinity for theprion rods as compared to 3F4 whereas all of the recombinant antibodiesbut for R1 show a lower degree of affinity against denatured antigen.

Example 18 Characterization Of Monoclonal Antibody ByImmunoprecipitation

[0207] Immunoprecipitation of SHaPrP 27-30 To confirm the anti-PrP 27-30activity of the Fabs as well as to confirm the in-ability of 3F4 to bindnondenatured SHaPrP 27-30, an immunoprecipitation method was developedusing liposomes containing SHa 27-30. E. coli extracts from Fabproducing clones immunoprecipitated 40-50% of the SHaPrP 27-30 presentin the solution, while 3F4 in dilution of {fraction (1/500)}immunoprecipitated only trace amounts of SHaPrP. Fab concentrations inbacterial supernates are typically on the order of 1-10 pg/ml. Thisimplies that the affinity for antigen are high (on the order of 10⁷-10⁸moles/liter or more). The antibody 3F4 was obtained as an ascetic fluidand is expected to have a concentration of approximately 1 μg/ml at thedilution used in the immunoprecipitation experiment. The ability of thenew Fabs to immunoprecipitate SHaPrP 27-30 in comparison to 3F4 wasdetermined quantitatively with purified Fab mAbs D4 and R2. Fab 2Rimmunoprecipitated SHaPrP 27-30 strongly at concentrations as low as 0.1pg/ml (50 ng in 500 pl) indicating an affinity on the order of greaterthan 10⁸M⁻¹ (i.e., 10⁸ mole/liter). Fab 2R was less potent but clearlyimmune precipitated antigen more efficiently than 3F4. Note that D4, R2,6D2, D14, R1, and R10 all refer to antibodies of the invention.

Immunoprecipitation of SHaPrP 27-30 with Recombinant Fabs

[0208] The ability of 3F4 diluted {fraction (1/500)} and 100 μl of E.Coli extracts containing Fab to immunoprecipitate SHaPrP 27-30 wasmonitored by western blotting. All lanes except lane 14 are fromimmunoprecipitations containing goat anti-mouse lgG Fab and protein Aagarose. 10 μl of liposomes containing SHa PrP 27-30 were added to lanes1, 3, 5, 7, 9, 11, 13. 100 μl of E. coli extracts from different clonesdiluted {fraction (1/500)} were added as follows: lanes 2-3, 6D2; lanes4-5, D14; lanes 6-7, R1; lanes 8-9, R10; lanes 10-11, D4; lanes 12-13,-3F4. Lane 14 was loaded with ½ volume of liposomes used forimmunoprecipitations.

[0209] The results described above are shown within the photograph ofFIG. 11. The photo clearly shows higher degrees of immunoprecipitationwhen using the recombinant antibodies of the invention.

[0210]FIG. 12 is a photo showing the immunoprecipitation of SHaPrP 27-30with purified Fabs of the invention (2R and 4D) as well as 3H4. Theability to immunoprecipitate the antigen is monitored by westernblotting. All of the lanes shown in FIG. 12 but for lane 14 areimmunoprecipitations containing goat anti-mouse IgG Fab and proteinAgarose. To obtain the results 10 μl of liposomes containing SHaPrP27-30 were added to all lanes except for lanes 5, 9 and 13. Each of thelanes are marked with the indicated amounts of purified Fabs (nanograms)which were added to lanes 2-13. Lane 14 was loaded with one-half volumeof liposomes used for the immunoprecipitation. The results clearly showa dramatically higher degree of precipitation when using the antibodies2R and 4D of the invention as compared to 3F4.

[0211] The ELISA data (FIG. 9) clearly show a number of Fabs with asaturable binding to non-denatured PrP 27-30 and a half-maximal bindingat around 0.5 μg/ml. This corresponds to an apparent affinity constantat 10⁸ M⁻¹ (MW of Fab=50,000). At the same time, 3F4 shows insignificantbinding out to 2 μg/ml. Moving to denatured PrP 27-30, FIG. 10, therecombinant Fabs now bind to a higher level but with a similar apparentaffinity. This suggests denaturation has revealed more antigenic sitesbut their affinities are the same. Significantly, 3F4 is now bindingcomparably to the recombinant Fabs with an apparent affinity of theorder of 10⁸ M⁻¹. Comparison of the 3F4 data in FIGS. 9 and 10 stronglysuggests the integrity of PrP 27-30 in the non-denatured form. Thus itcould have been argued that the recombinant Fabs were reacting with afraction of denatured PrP present in the PrP 27-30 preparation. The lackof reactivity of 3F4 with non-denatured PrP 27-30 coupled with itsstrong reactivity with denatured PrP 27-30 refutes this interpretationand strongly suggests the recombinant Fabs recognize non-denatured rodswith high affinity.

[0212] The immunoprecipitation data are confirmatory of the ELISA-data.Low concentrations of recombinant Fabs as found in crude bacterialsupernates (typically 1-10 μl/ml) are highly effective atimmunoprecipitating PrP 27-30 (FIG. 11). This implies an affinity on theorder of 10⁷-10⁸ M⁻¹. Under comparable concentration conditions, 3F4does not produce significant precipitation. A more quantitative analysis(FIG. 12) shows that Fab R2 immunoprecipitates PrP 27-30 highlyeffectively with some titration in the range 0.1-0.2 μg/ml implying abinding affinity on the order of 10⁸ M⁻¹. Fab 4D has a lower affinityand 3F4 immunoprecipitates very weakly indeed. From this-particularexperiment one could argue that the affinity of 3F4 is considerably lessthan 5×10⁷ M⁻¹ and probably less than 10⁷ M⁻¹.

[0213] Overall, the data indicates that the recombinant Fabs haveaffinities in the range of 10⁷-10⁸ M⁻¹.

[0214] The instant invention is shown and described herein in what isconsidered to be a most practical and preferred embodiments. It isrecognized, however, that departures may be made from which are withinthe scope of the invention and that modifications will occur to one whois skilled in the art upon reading this disclosure.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 86 <210> SEQ ID NO 1<211> LENGTH: 254 <212> TYPE: PRT <213> ORGANISM: mouse <400> SEQUENCE:1 Met Ala Asn Leu Gly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp 1 5 1015 Thr Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 2530 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 4045 Tyr Pro Pro Gln Gly Gly Thr Trp Gly Gln Pro His Gly Gly Gly Trp 50 5560 Gly Gln Pro His Gly Gly Ser Trp Gly Gln Pro His Gly Gly Ser Trp 65 7075 80 Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His Asn 8590 95 Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Leu Lys His Val Ala100 105 110 Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly TyrMet 115 120 125 Leu Gly Ser Ala Met Ser Arg Pro Met Ile His Phe Gly AsnAsp Trp 130 135 140 Glu Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr ProAsn Gln Val 145 150 155 160 Tyr Tyr Arg Pro Val Asp Gln Tyr Ser Asn GlnAsn Asn Phe Val His 165 170 175 Asp Cys Val Asn Ile Thr Ile Lys Gln HisThr Val Thr Thr Thr Thr 180 185 190 Lys Gly Glu Asn Phe Thr Glu Thr AspVal Lys Met Met Glu Arg Val 195 200 205 Val Glu Gln Met Cys Val Thr GlnTyr Gln Lys Glu Ser Gln Ala Tyr 210 215 220 Tyr Asp Gly Arg Arg Ser SerSer Thr Val Leu Phe Ser Ser Pro Pro 225 230 235 240 Val Ile Leu Leu IleSer Phe Leu Ile Phe Leu Ile Val Gly 245 250 <210> SEQ ID NO 2 <211>LENGTH: 253 <212> TYPE: PRT <213> ORGANISM: homo sapien <400> SEQUENCE:2 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp 1 5 1015 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn 20 2530 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg 35 4045 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 50 5560 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly 65 7075 80 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His 8590 95 Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His Met100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly GlyTyr 115 120 125 Met Leu Gly Ser Ala Met Ser Arg Pro Ile Ile His Phe GlySer Asp 130 135 140 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met His Arg TyrPro Asn Gln 145 150 155 160 Val Tyr Tyr Arg Pro Met Asp Glu Tyr Ser AsnGln Asn Asn Phe Val 165 170 175 His Asp Cys Val Asn Ile Thr Ile Lys GlnHis Thr Val Thr Thr Thr 180 185 190 Thr Lys Gly Glu Asn Phe Thr Glu ThrAsp Val Lys Met Met Glu Arg 195 200 205 Val Val Glu Gln Met Cys Ile ThrGln Tyr Glu Arg Glu Ser Gln Ala 210 215 220 Tyr Tyr Gln Arg Gly Ser SerMet Val Leu Phe Ser Ser Pro Pro Val 225 230 235 240 Ile Leu Leu Ile SerPhe Leu Ile Phe Leu Ile Val Gly 245 250 <210> SEQ ID NO 3 <211> LENGTH:263 <212> TYPE: PRT <213> ORGANISM: bovine <400> SEQUENCE: 3 Met Val LysSer His Ile Gly Ser Trp Ile Leu Val Leu Phe Val Ala 1 5 10 15 Met TrpSer Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 20 25 30 Trp AsnThr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly 35 40 45 Asn ArgTyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His Gly 50 55 60 Gly GlyTrp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 65 70 75 80 GlyGly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly 85 90 95 GlyGly Gly Trp Gly Gln Gly Gly Thr His Gly Gln Trp Asn Lys Pro 100 105 110Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala Ala Ala Ala 115 120125 Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met 130135 140 Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu Asp Arg Tyr Tyr145 150 155 160 Arg Glu Asn Met His Arg Tyr Pro Asn Gln Val Tyr Tyr ArgPro Val 165 170 175 Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val His Asp CysVal Asn Ile 180 185 190 Thr Val Lys Glu His Thr Val Thr Thr Thr Thr LysGly Glu Asn Phe 195 200 205 Thr Glu Thr Asp Ile Lys Met Met Glu Arg ValVal Glu Gln Met Cys 210 215 220 Val Thr Gln Tyr Gln Lys Glu Ser Gln AlaTyr Tyr Asp Gln Gly Ala 225 230 235 240 Ser Val Ile Leu Phe Ser Ser ProPro Val Ile Leu Leu Ile Ser Phe 245 250 255 Leu Ile Phe Leu Ile Val Gly260 <210> SEQ ID NO 4 <211> LENGTH: 255 <212> TYPE: PRT <213> ORGANISM:ovine <400> SEQUENCE: 4 Met Val Lys Ser His Ile Gly Ser Trp Ile Leu ValLeu Phe Val Ala 1 5 10 15 Met Trp Ser Asp Val Gly Leu Cys Lys Lys ArgPro Lys Pro Gly Gly 20 25 30 Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly GlnGly Ser Pro Gly Gly 35 40 45 Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly TrpGly Gln Pro His Gly 50 55 60 Gly Gly Trp Gly Gln Pro His Gly Gly Gly TrpGly Gln Pro His Gly 65 70 75 80 Gly Ser Trp Gly Gln Pro His Gly Gly GlyGly Trp Gly Gln Gly Gly 85 90 95 Ser His Ser Gln Trp Asn Lys Pro Ser LysPro Lys Thr Asn Met Lys 100 105 110 His Val Ala Gly Ala Ala Ala Ala GlyAla Val Val Gly Gly Leu Gly 115 120 125 Gly Tyr Met Leu Gly Ser Ala MetSer Arg Pro Leu Ile His Phe Gly 130 135 140 Asn Asp Tyr Glu Asp Arg TyrTyr Arg Glu Asn Met Tyr Arg Tyr Pro 145 150 155 160 Asn Gln Val Tyr TyrArg Pro Val Asp Gln Tyr Ser Asn Gln Asn Asn 165 170 175 Phe Val His AspCys Val Asn Ile Thr Val Lys Gln His Thr Val Thr 180 185 190 Thr Thr ThrLys Gly Glu Asn Phe Thr Glu Thr Asp Ile Lys Ile Met 195 200 205 Glu ArgVal Val Glu Gln Met Cys Ile Thr Gln Tyr Gln Arg Glu Ser 210 215 220 GlnAla Tyr Tyr Gln Arg Gly Ala Ser Val Ile Leu Phe Ser Ser Pro 225 230 235240 Pro Val Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 245 250255 <210> SEQ ID NO 5 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:mouse <400> SEQUENCE: 5 caggtgcagc tcgaggagtc aggacct 27 <210> SEQ ID NO6 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE:6 gaggtgcagc tcgaggagtc aggacct 27 <210> SEQ ID NO 7 <211> LENGTH: 27<212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 7 gaggtccagctcgagcagtc tggacct 27 <210> SEQ ID NO 8 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: mouse <400> SEQUENCE: 8 caggtccaac tcgagcagcc tggggtc 27<210> SEQ ID NO 9 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: mouse<400> SEQUENCE: 9 gaggttcagc tcgagcagtc tggggcaa 28 <210> SEQ ID NO 10<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE:10 gaagtgaagc tcgaggagtc tggagga 27 <210> SEQ ID NO 11 <211> LENGTH: 27<212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 11 gaggtgaagctcgaggagtc tggagga 27 <210> SEQ ID NO 12 <211> LENGTH: 27 <212> TYPE:DNA <213> ORGANISM: mouse <400> SEQUENCE: 12 gaggtgaagc ttctcgagtctggaggt 27 <210> SEQ ID NO 13 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 13 gaagtgaagc tcgaggagtc tggggga 27<210> SEQ ID NO 14 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:mouse <400> SEQUENCE: 14 gaggttcagc tcgaggagca gtctggagct 30 <210> SEQID NO 15 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 15 aggtccagct gctcgagtct gg 22 <210> SEQ ID NO 16 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 16aggtgcagct gctcgagtct gg 22 <210> SEQ ID NO 17 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 17 aggtcaagct gctcgagtctgg 22 <210> SEQ ID NO 18 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 18 aggtgaagct gctcgagtct gg 22 <210> SEQID NO 19 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 19 aggtccaact gctcgagtct gg 22 <210> SEQ ID NO 20 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 20aggtgcaact gctcgagtct gg 22 <210> SEQ ID NO 21 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 21 aggtcaaact gctcgagtctgg 22 <210> SEQ ID NO 22 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 22 aggtgaaact gctcgagtct gg 22 <210> SEQID NO 23 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 23 aggtccagct tctcgagtct gg 22 <210> SEQ ID NO 24 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 24aggtgcagct tctcgagtct gg 22 <210> SEQ ID NO 25 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 25 aggtcaagct tctcgagtctgg 22 <210> SEQ ID NO 26 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 26 aggtgaagct tctcgagtct gg 22 <210> SEQID NO 27 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 27 aggtccaact tctcgagtct gg 22 <210> SEQ ID NO 28 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 28aggtgcaact tctcgagtct gg 22 <210> SEQ ID NO 29 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 29 aggtcaaact tctcgagtctgg 22 <210> SEQ ID NO 30 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 30 aggtgaaact tctcgagtct gg 22 <210> SEQID NO 31 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 31 aggtccagct gctcgagtca gg 22 <210> SEQ ID NO 32 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 32aggtgcagct gctcgagtca gg 22 <210> SEQ ID NO 33 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 33 aggtcaagct gctcgagtcagg 22 <210> SEQ ID NO 34 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 34 aggtgaagct gctcgagtca gg 22 <210> SEQID NO 35 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 35 aggtccaact gctcgagtca gg 22 <210> SEQ ID NO 36 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 36aggtgcaact gctcgagtca gg 22 <210> SEQ ID NO 37 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 37 aggtcaaact gctcgagtcagg 22 <210> SEQ ID NO 38 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 38 aggtgaaact gctcgagtca gg 22 <210> SEQID NO 39 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 39 aggtccagct tctcgagtca gg 22 <210> SEQ ID NO 40 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 40aggtgcagct tctcgagtca gg 22 <210> SEQ ID NO 41 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 41 aggtcaagct tctcgagtcagg 22 <210> SEQ ID NO 42 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 42 aggtgaagct tctcgagtca gg 22 <210> SEQID NO 43 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 43 aggtccaact tctcgagtca gg 22 <210> SEQ ID NO 44 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 44aggtgcaact tctcgagtca gg 22 <210> SEQ ID NO 45 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 45 aggtcaaact tctcgagtcagg 22 <210> SEQ ID NO 46 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 46 aggtgaaact tctcgagtca gg 22 <210> SEQID NO 47 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 47 aggtccagct gctcgagtct gg 22 <210> SEQ ID NO 48 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 48aggtccagct gctcgagtca gg 22 <210> SEQ ID NO 49 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 49 aggtccagct tctcgagtctgg 22 <210> SEQ ID NO 50 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 50 aggtccagct tctcgagtca gg 22 <210> SEQID NO 51 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 51 ccagttccga gctcgttgtg actcaggaat ct 32 <210> SEQ ID NO 52<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE:52 ccagttccga gctcgtggtg acgcagccgc cc 32 <210> SEQ ID NO 53 <211>LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 53ccagttccga gctcgtgctc acccagtctc ca 32 <210> SEQ ID NO 54 <211> LENGTH:32 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 54 ccagttccgagctccagatg acccagtctc ca 32 <210> SEQ ID NO 55 <211> LENGTH: 29 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 55 ccagatgtga gctcgtgacccagactcca 29 <210> SEQ ID NO 56 <211> LENGTH: 32 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 56 ccagatgtga gctcgtcatg acccagtctc ca32 <210> SEQ ID NO 57 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:mouse <400> SEQUENCE: 57 ccagttccga gctcgtgatg acacagtctc ca 32 <210>SEQ ID NO 58 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: mouse<400> SEQUENCE: 58 gcgccgtcta gaattaacac tcattcctgt tgaa 34 <210> SEQ IDNO 59 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 59 aggtccaact gctcgagtct gg 22 <210> SEQ ID NO 60 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 60aggtccaact gctcgagttc ag 22 <210> SEQ ID NO 61 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 61 aggtccaact tctcgagtctgg 22 <210> SEQ ID NO 62 <211> LENGTH: 30 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 62 aggcttacta gtacaatccc tgggcacaat 30<210> SEQ ID NO 63 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:mouse <400> SEQUENCE: 63 ctccttacta gtaggacagg ggattgt 27 <210> SEQ IDNO 64 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: mouse <400>SEQUENCE: 64 cacgactgag gcacctcc 18 <210> SEQ ID NO 65 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: mouse <400> SEQUENCE: 65 aagacagctatcgcgattgc ag 22 <210> SEQ ID NO 66 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: mouse <400> SEQUENCE: 66 atagcccttg accaggcatcccagggtcac 30 <210> SEQ ID NO 67 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: mouse <400> SEQUENCE: 67 acctattgcc tacggcagcc g 21 <210> SEQID NO 68 <211> LENGTH: 114 <212> TYPE: PRT <213> ORGANISM: mouse <400>SEQUENCE: 68 Leu Glu Gln Ser Gly Val Glu Leu Ala Arg Pro Gly Ala Ser ValMet 1 5 10 15 Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr GlyIle Ser 20 25 30 Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile GlyGlu Ile 35 40 45 Trp Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe LysGly Lys 50 55 60 Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr LeuAsp Leu 65 70 75 80 Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe CysAla Arg His 85 90 95 Asp Gly Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr LeuVal Thr Val 100 105 110 Ser Ala <210> SEQ ID NO 69 <211> LENGTH: 114<212> TYPE: PRT <213> ORGANISM: mouse <400> SEQUENCE: 69 Leu Glu Gln SerGly Val Glu Leu Ala Arg Pro Gly Ala Ser Val Met 1 5 10 15 Leu Ser CysLys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Gly Ile Ser 20 25 30 Trp Val LysGln Arg Thr Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile 35 40 45 Cys Pro ArgSer Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys 50 55 60 Ala Thr LeuThr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu Asp Leu 65 70 75 80 Arg SerLeu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg His 85 90 95 Asp GlyTyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 SerAla <210> SEQ ID NO 70 <211> LENGTH: 91 <212> TYPE: PRT <213> ORGANISM:mouse <400> SEQUENCE: 70 Tyr Thr Phe Thr Thr Tyr Gly Ile Thr Trp Val LysGln Arg Thr Gly 1 5 10 15 Gln Gly Leu Glu Trp Ile Gly Glu Ile Trp ProArg Ser Gly Asn Thr 20 25 30 Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala ThrLeu Thr Ala Asp Lys 35 40 45 Ser Ser Ser Thr Ala Tyr Met Glu Val Arg SerLeu Thr Ser Asp Asp 50 55 60 Ser Ala Val Tyr Phe Cys Ala Arg His Asp GlyTyr Pro Phe Ala Tyr 65 70 75 80 Trp Gly Gln Gly Thr Leu Val Thr Val SerAla 85 90 SEQ ID NO 71 <211> LENGTH: 91 <212> TYPE: PRT <213> ORGANISM:mouse <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 1 <223>OTHER INFORMATION: Xaa = Any Amino Acid <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: 1 <223> OTHER INFORMATION: Xaa = Any Amino Acid<400> SEQUENCE: 71 Xaa Thr Phe Thr Val Tyr Gly Ile Ser Trp Val Lys GlnArg Thr Gly 1 5 10 15 Gln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro ArgSer Gly Asn Thr 20 25 30 Tyr Tyr Asn Glu Lys Phe Lys Val Lys Ala Thr LeuSer Ala Asp Lys 35 40 45 Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser LeuThr Ser Glu Asp 50 55 60 Ser Ala Val Tyr Phe Cys Ala Arg His Asp Gly TyrPro Phe Ala Tyr 65 70 75 80 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala85 90 SEQ ID NO 72 <211> LENGTH: 95 <212> TYPE: PRT <213> ORGANISM:mouse <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 3 <223>OTHER INFORMATION: Xaa = Any Amino Acid <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: 3 <223> OTHER INFORMATION: Xaa = Any Amino Acid<400> SEQUENCE: 72 Trp Glu Xaa Arg Val Ser Leu Thr Cys Arg Ala Ser GlnAsp Phe Gly 1 5 10 15 Ser Ser Leu Asn Trp Phe Arg Gln Lys Pro Asp GlyThr Ile Arg Arg 20 25 30 Leu Ile Tyr Ala Thr Ser Arg Leu His Ser Gly ValPro Lys Arg Phe 35 40 45 Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu ThrIle Ser Ser Leu 50 55 60 Glu Ala Glu Asp Phe Gly Asp Tyr Tyr Cys Leu GlnTyr Ala Ala Ser 65 70 75 80 Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu GluIle Lys Arg Ala 85 90 95 SEQ ID NO 73 <211> LENGTH: 109 <212> TYPE: PRT<213> ORGANISM: mouse <400> SEQUENCE: 73 Glu Leu Val Met Thr Gln Thr ProSer Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr CysArg Ala Ser Gln Asp Phe Gly Ser Ser 20 25 30 Leu Asn Trp Phe Arg Gln AlaPro Asp Gly Thr Ile Arg Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Lys Leu HisSer Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp HisSer Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Leu Gly Asn TyrTyr Cys Leu Gln Tyr Ala Ala Ser Pro Phe 85 90 95 Thr Phe Gly Ser Gly ThrLys Leu Glu Ile Lys Arg Ala 100 105 <210> SEQ ID NO 74 <211> LENGTH: 109<212> TYPE: PRT <213> ORGANISM: mouse <400> SEQUENCE: 74 Glu Leu Gln MetThr Gln Thr Pro Ser Ser Leu Ser Val Ser Leu Gly 1 5 10 15 Glu Arg ValSer Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser 20 25 30 Leu Asn TrpLeu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala ThrSer Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg SerGly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu AspLeu Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Trp 85 90 95 Thr PheGly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 105 <210> SEQ ID NO 75<211> LENGTH: 105 <212> TYPE: PRT <213> ORGANISM: mouse <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: 1, 14 <223> OTHER INFORMATION:Xaa = Any Amino Acid <220> FEATURE: <221> NAME/KEY: VARIANT <222>LOCATION: 1, 14 <223> OTHER INFORMATION: Xaa = Any Amino Acid <400>SEQUENCE: 75 Xaa Leu Gly Arg Gln Val Met Leu Ser Ser Lys Ala Ser Xaa TyrThr 1 5 10 15 Phe Thr Thr Tyr Gly Ile Ser Trp Val Lys Gln Arg Thr GlyGln Gly 20 25 30 Leu Glu Trp Ile Gly Glu Ile Cys Pro Arg Ser Gly Asn ThrTyr Tyr 35 40 45 Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp LysSer Ser 50 55 60 Ser Thr Ala Tyr Leu Asp Leu Arg Ser Leu Thr Ser Glu AspSer Ala 65 70 75 80 Val Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe AlaTyr Trp Gly 85 90 95 Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 <210>SEQ ID NO 76 <211> LENGTH: 114 <212> TYPE: PRT <213> ORGANISM: mouse<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 13 <223> OTHERINFORMATION: Xaa = Any Amino Acid <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: 13 <223> OTHER INFORMATION: Xaa = Any Amino Acid <400>SEQUENCE: 76 Leu Glu Gln Ser Gly Val Glu Leu Ala Arg Pro Gly Xaa Ser ValLys 1 5 10 15 Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr GlyIle Thr 20 25 30 Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile GlyGlu Ile 35 40 45 Trp Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe LysGly Lys 50 55 60 Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr MetGlu Val 65 70 75 80 Arg Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe CysAla Arg His 85 90 95 Asp Gly Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr LeuVal Thr Val 100 105 110 Ser Ala <210> SEQ ID NO 77 <211> LENGTH: 114<212> TYPE: PRT <213> ORGANISM: mouse <400> SEQUENCE: 77 Leu Glu Gln SerGly Val Glu Leu Ala Gly Pro Gly Ala Ser Val Lys 1 5 10 15 Leu Ser CysLys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr Gly Ile Ser 20 25 30 Trp Val LysGln Arg Thr Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile 35 40 45 Trp Pro ArgSer Gly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys 50 55 60 Ala Thr LeuThr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Leu Asp Leu 65 70 75 80 Arg SerLeu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg His 85 90 95 Asp GlyTyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 SerAla <210> SEQ ID NO 78 <211> LENGTH: 91 <212> TYPE: PRT <213> ORGANISM:mouse <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 1 <223>OTHER INFORMATION: Xaa = Any Amino Acid <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: 1 <223> OTHER INFORMATION: Xaa = Any Amino Acid<400> SEQUENCE: 78 Xaa Thr Phe Thr Thr Tyr Gly Ile Thr Trp Val Lys GlnArg Thr Gly 1 5 10 15 Gln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro ArgSer Gly Asn Thr 20 25 30 Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr LeuThr Ala Asp Lys 35 40 45 Ser Ser Ser Thr Ala Tyr Met Glu Val Arg Ser LeuThr Ser Asp Asp 50 55 60 Ser Ala Val Tyr Phe Cys Ala Arg His Asp Gly TyrPro Phe Ala Tyr 65 70 75 80 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala85 90 SEQ ID NO 79 <211> LENGTH: 92 <212> TYPE: PRT <213> ORGANISM:mouse <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 1 <223>OTHER INFORMATION: Xaa = Any Amino Acid <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: 1 <223> OTHER INFORMATION: Xaa = Any Amino Acid<400> SEQUENCE: 79 Xaa Tyr Thr Phe Thr Thr Tyr Gly Ile Thr Trp Val LysGln Arg Thr 1 5 10 15 Gly Gln Asp Leu Glu Trp Ile Gly Glu Ile Trp ProArg Ser Gly Asn 20 25 30 Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala ThrLeu Ala Ala Asp 35 40 45 Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg SerLeu Thr Ser Asp 50 55 60 Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp GlyTyr Pro Phe Ala 65 70 75 80 Tyr Trp Asp Gln Gly Thr Leu Val Thr Val SerThr 85 90 SEQ ID NO 80 <211> LENGTH: 99 <212> TYPE: PRT <213> ORGANISM:mouse <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 1 <223>OTHER INFORMATION: Xaa = Any Amino Acid <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: 1 <223> OTHER INFORMATION: Xaa = Any Amino Acid<400> SEQUENCE: 80 Xaa Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr ValTyr Gly Ile 1 5 10 15 Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu GluTrp Ile Gly Glu 20 25 30 Ile Trp Pro Arg Ser Gly Asn Thr Tyr Tyr Asn GluLys Phe Lys Val 35 40 45 Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser ThrAla Ser Met Glu 50 55 60 Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val TyrPhe Cys Ala Arg 65 70 75 80 His Asp Gly Tyr Pro Phe Ala Tyr Trp Gly GlnGly Thr Leu Val Thr 85 90 95 Val Ser Ala <210> SEQ ID NO 81 <211>LENGTH: 91 <212> TYPE: PRT <213> ORGANISM: mouse <220> FEATURE: <221>NAME/KEY: VARIANT <222> LOCATION: 1, 46 <223> OTHER INFORMATION: Xaa =Any Amino Acid <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 1,46 <223> OTHER INFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 81 XaaThr Phe Thr Val Tyr Gly Ile Ser Trp Val Lys Gln Arg Thr Gly 1 5 10 15Gln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg Ser Gly Asn Thr 20 25 30Tyr Tyr Asn Glu Lys Phe Lys Val Lys Ala Thr Leu Thr Xaa Asp Lys 35 40 45Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp 50 55 60Ser Ala Val Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe Ala Tyr 65 70 7580 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Thr 85 90 SEQ ID NO 82 <211>LENGTH: 101 <212> TYPE: PRT <213> ORGANISM: mouse <400> SEQUENCE: 82 SerVal Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 1 5 10 15Gly Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 20 25 30Gly Glu Ile Trp Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 35 40 45Lys Gly Lys Ala Thr Leu Ser Ala Asp Lys Ser Ser Ser Thr Ala Tyr 50 55 60Leu Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 65 70 7580 Ala Arg His Asp Gly Tyr Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu 85 9095 Val Thr Val Ser Ala 100 <210> SEQ ID NO 83 <211> LENGTH: 108 <212>TYPE: PRT <213> ORGANISM: mouse <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: 3, 4, 18 <223> OTHER INFORMATION: Xaa = Any Amino Acid<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 3, 4, 18 <223>OTHER INFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 83 Glu Leu XaaXaa Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser 1 5 10 15 Gly XaaThr Phe Thr Thr Tyr Gly Ile Thr Trp Val Lys Gln Arg Thr 20 25 30 Gly GlnGly Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg Ser Gly Asn 35 40 45 Thr TyrTyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp 50 55 60 Lys SerSer Ser Thr Ala Tyr Met Glu Val Arg Ser Leu Thr Ser Asp 65 70 75 80 AspSer Ala Val Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe Ala 85 90 95 TyrTrp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 <210> SEQ ID NO 84<211> LENGTH: 103 <212> TYPE: PRT <213> ORGANISM: mouse <400> SEQUENCE:84 Pro Gly Pro Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 1 510 15 Thr Thr Tyr Gly Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu 2025 30 Glu Trp Ile Gly Glu Ile Trp Pro Arg Ser Gly Asn Thr Tyr Tyr Asn 3540 45 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 5055 60 Thr Ala Tyr Leu Asp Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val 6570 75 80 Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe Ala Tyr Trp Gly Gln85 90 95 Gly Thr Leu Val Thr Val Ser 100 <210> SEQ ID NO 85 <211>LENGTH: 92 <212> TYPE: PRT <213> ORGANISM: mouse <220> FEATURE: <221>NAME/KEY: VARIANT <222> LOCATION: 1 <223> OTHER INFORMATION: Xaa = AnyAmino Acid <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 1<223> OTHER INFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 85 Xaa AsnThr Phe Thr Thr Tyr Gly Ile Ser Trp Val Lys Gln Arg Thr 1 5 10 15 GlyGln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg Ser Gly Asn 20 25 30 ThrTyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp 35 40 45 LysSer Ser Ser Thr Ala Tyr Leu Asp Leu Arg Ser Leu Thr Ser Glu 50 55 60 AspSer Ala Val Tyr Phe Cys Ala Arg His Asp Gly Tyr Pro Phe Ala 65 70 75 80Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 85 90 SEQ ID NO 86 <211>LENGTH: 95 <212> TYPE: PRT <213> ORGANISM: mouse <220> FEATURE: <221>NAME/KEY: VARIANT <222> LOCATION: 1 <223> OTHER INFORMATION: Xaa = AnyAmino Acid <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 1<223> OTHER INFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 86 Xaa AlaSer Gly Tyr Thr Phe Thr Thr Tyr Gly Ile Ser Trp Val Lys 1 5 10 15 GlnArg Thr Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile Trp Pro Arg 20 25 30 SerGly Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu 35 40 45 ThrAla Asp Lys Ser Ser Ser Thr Ala Tyr Leu Asp Leu Arg Ser Leu 50 55 60 ThrSer Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Gly Tyr 65 70 75 80Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 85 90 95

1. An antibody characterized by its ability to bind to PrP^(Sc) in situ.2. The antibody of claim 1, wherein the antibody specifically binds toPrP^(Sc) of a mammal selected from the group consisting of a human, acow, a sheep, a horse, a pig, a dog, a chicken and a cat and furtherwherein the antibody specifically binds to human PrP^(Sc) in a nativenon-denatured form.
 3. The antibody of claim 1, wherein the antibodybinds to PrP^(Sc) with a binding affinity K_(a) of 10⁷ l/mol or more. 4.The antibody of claim 3, wherein the K_(a) is 10⁸ l/mole or more.
 5. Theantibody of claim 1, further characterized by the ability of theantibody to neutralize prion protein infectivity and have a K_(a) of 10⁸l/mole or more.
 6. An antibody which specifically binds to PrP^(Sc),produced by the process comprising the steps of: (a) synthesizing alibrary of antibodies on phage; (b) panning the library against a sampleby bringing the phage into contact with a composition comprising PrPproteins; (c) isolating phage which bind PrP^(Sc) protein.
 7. Theantibody of claim 6, further comprising: (d) analyzing the phage of (c)to determine a sequence encoding an amino acid sequence to which theprion protein of (c) binds.
 8. The antibody of claim 6, wherein thelibrary of antibodies on phage are prepared by: (1a) immunizing a hostmammal with PrP protein to create an immune response; (2a) extractingcells from the host mammal which cells are responsible for production ofantibodies; (3a) isolating RNA from the cells of (2a); (4a) reversetranscribing the PNA to produce cDNA; (5a) amplifying the cDNA using aprimer; and (6a) inserting the cDNA of (5a) into a phage display vectorsuch that antibodies are expressed on the phage.
 9. The antibody ofclaim 6, further comprising: panning antibodies against an antigendispersed in a liposome.
 10. The antibody of claim 9, wherein theantigen dispersed in a liposome is PrP^(Sc).
 11. The antibody of claim9, wherein the antigen dispersed in a liposome is a core portion ofPrP^(Sc) not digested with proteinase K which core portion isbiotinylated.
 12. A method of detecting human PrP^(Sc) in a sourcecomprising: contacting a source suspected of containing human PrP^(Sc)with a diagnostically effective amount of an antibody which specificallybinds 50% or more of human PrP^(Sc) in the source; and, determiningwhether the antibody binds specifically to any material in the source.13. The method of claim 12, wherein the antibody is attached to adetectable label and the detecting is in vivo.
 14. The method of claim13 wherein the label is selected from a group consisting of aradioisotope label and a paramagnetic label; and wherein the antibody isattached to a substrate and the detecting is in vitro.
 15. An assay,comprising: a support surface; and an antibody bound to the surface ofthe support, the antibody characterized by an ability to bind PrP^(Sc)in situ with a binding affinity of 10⁷ l/mole or more.
 16. The asay ofclaim 15, wherein the antibody is characterized by an ability to bind50% or more of PrP^(Sc) in a liquid flowable sample.
 17. The assay ofclaim 15, wherein a plurality of different antibodies are bound to thesupport surface and each antibody has a K_(a) of 10⁷ l/mole or morerelative to PrP^(Sc).
 18. A method of determining the cause of death ofan animal, comprising: extracting tissue from an animal that has died;contacting the tissue with an antibody of claim 1 wherein the antibodybinds to a form of PrP^(Sc) specific to the animal that has died; anddetermining if the antibody has bound to PrP^(Sc).
 19. A method ofpurifying a material suspected of containing a PrP^(Sc) protein,comprising: contacting the material with a sufficient amount of anantibody of claim 1 which antibody is bound to a support surface, andremoving material not bound to the antibody.
 20. A method of treating amaterial, comprising adding to the material a sufficient amount of anantibody of claim 1 to neutralize PrP^(Sc) protein infectivity.